Influenza treatment and/or characterization, human-adapted HA polypeptides; vaccines

ABSTRACT

The present invention provides, among other things, methods, reagents, and systems for the treatment, detection, analysis, and/or characterization of influenza infections.

RELATED APPLICATIONS

This patent application claims priority to U.S. provisional PatentApplication Ser. No. 61/384,780, filed Sep. 21, 2010, the disclosure ofwhich is incorporated herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. R37GM057073 and U54 GM2116 awarded by the National Institutes of Health.The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Influenza has a long history of pandemics, epidemics, resurgences andoutbreaks. Among influenza strains, H2 strains pose particularchallenges in light of the waning population immunity to H2hemagglutinin. There is a need for vaccines and therapeutic strategiesfor effective treatment or delay of onset of disease caused by influenzavirus; there is a particular need for vaccines and therapeuticstrategies for effective treatment or delay of onset of disease causedby H2 influenza viruses.

SUMMARY OF THE INVENTION

The present invention provides binding agents that show a strong abilityto discriminate between umbrella-topology and cone-topology glycans. Insome embodiments, provided binding agents are engineered HApolypeptides. In some embodiments, provided binding agents areengineered H2 HA polypeptides. In some embodiments, provided bindingagents show an ability to discriminate between umbrella-topology andcone-topology glycans that is at least effective as that shown by anRTLS HA polypeptide (e.g., an RTLS H2 HA polypeptide) as describedherein.

In some embodiments, the present invention provides, among other things,engineered hemagglutinin (HA) polypeptides that include a sequenceelement referred to herein as “RTLS”. As described herein, the RTLSelement refers to the presence of particular amino acids at positionscorresponding to residues 137, 193, 226, and 228 of the HA polypeptide.In some embodiments, the present invention provides improvements toengineered HA polypeptides, for example in that the improved engineeredHA polypeptides have certain particular amino acids residues atpositions corresponding to 137, 193, 226, and 228. As described herein,such HA polypeptides have a variety of unexpected and usefulcharacteristics as compared with prior art HA polypeptides, includingprior art engineered HA polypeptides.

The present invention also provides, for example, diagnostic andtherapeutic reagents and methods associated with provided bindingagents, including vaccines. Among other things, provided reagents andmethods are useful in the practice of medicine, for example in thedelivery of vaccines and/or for the treatment or prevention ofinfection, for example with the influenza virus. In some embodiments,provided reagents and methods are particularly useful in the treatmentof humans. In some embodiments, the present invention providesimprovements to certain diagnostic and/or therapeutic reagents andmethods, which improvement comprises, for example, inclusion,preparation and/or use of an engineered HA polypeptide as describedherein, and/or of an HA polypeptide having certain particular aminoacids residues at positions corresponding to 137, 193, 226, and 228.

The present invention also provides, for example, systems and reagentsfor identifying binding agents that effectively discriminate betweenumbrella-topology and cone-topology glycans. In some embodiments, suchbinding agents show at least as strong an ability to discriminate asdoes an RTLS HA polypeptide (e.g., an RTLS H2 HA polypeptide) asdescribed herein. In some embodiments, provided binding agents showenhanced binding to umbrella-topology glycans as compared with aparticular reference. In some embodiments, provided binding agents showreduced binding to cone-topology glycans as compared with a particularreference. In some embodiments, provided binding agents show bothenhanced binding to umbrella-topology glycans and reduced ability tocone-topology glycans as compared with a particular reference. In someembodiments, the particular reference is a wild-type HA polypeptide. Insome embodiments the particular reference is a wild-type H2 HApolypeptide. In some embodiments the particular reference is an RTLS HApolypeptide (e.g., an RTLS H2HA polypeptide). In some embodiments, thepresent invention provides improved systems and/or methods foridentifying desirable binding agents, wherein the improvement comprisesuse (e.g., comparison with) of an HA polypeptide (e.g., an engineeredpolypeptide) having certain particular amino acids residues at positionscorresponding to 137, 193, 226, and 228.

In some embodiments, provided binding agents (including provided HApolypeptides, e.g., engineered HA polypeptides) show an affinity (Kd′)for umbrella-topology glycans within the range of about 1.5 nM to about2 pM. In some embodiments, provided binding agents show an affinity(Kd′) for umbrella-topology glycans within the range of about 1.5 nM toabout 200 pM. In some embodiments, provided binding agents show anaffinity (Kd′) for umbrella-topology glycans within the range of about200 pM to about 10 pM. In some embodiments, provided binding agents showan affinity (Kd′) for umbrella-topology glycans within the range ofabout 10 pM to about 2 pM. In some embodiments, provided binding agentsshow an affinity (Kd′) for cone-topology glycans that is not less than 2nM; in some embodiments, provided binding agents show an affinity (Kd′)for cone-topology glycans that is within the range of about 200 pM toabout 2 nM. In some embodiments, provided binding agents show a relativeaffinity for umbrella glycans vs cone glycans that is about 1, about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 20, about 30, about 40, about 50, about 60, about 70, about 80,about 90, about 100, about 200, about 300, about 400, about 500, about600, about 700, about 800, about 900, about 1000, about 2000, about3000, about 4000, about 5000, about 6000, about 7000, about 8000, about9000, about 10,000, up to about 100,000 or more. In some embodiments,inventive binding agents show an affinity for umbrella topology glycansthat is about 100%, about 200%, about 300%, about 400%, about 500%,about 600%, about 700%, about 800%, about 900%, about 1000%, about2000%, about 3000%, about 4000%, about 5000%, about 6000%, about 7000%,about 8000%, about 9000%, about 10,000% or more than their affinity forcone topology glycans.

In some embodiments, the present invention provides HA polypeptides(e.g., engineered HA polypeptides) whose amino acid sequence includes anelement as set forth below:

-   -   X137 L1 X193 L2 X226 L3 X228 (SEQ ID NO. 28), wherein:    -   X137 is an amino acid selected from the group consisting of        arginine, lysine, glutamine, methionine and histidine; in some        embodiments, X137 is selected from the group consisting of        arginine and lysine; in some embodiments, X137 arginine;    -   L1 is a linker comprising approximately 40-70 amino acids;    -   X193 is an amino acid selected from the group consisting of        alanine, aspartic acid, glutamic acid, leucine, isoleucine,        methionine, serine, threonine, cysteine, and valine; in some        embodiments, X193 is selected from the group consisting of        alanine, glutamic acid and threonine; in some embodiments, X193        is threonine;    -   L2 is a linker comprising approximately 20-50 amino acids;    -   X226 is an amino acid selected from the group consisting of        alanine, cysteine, glycine, isoleucine, leucine, methionine,        phenylalanine, proline, tryptophan, and valine; in some        embodiments, X226 is selected from the group consisting of        leucine, isoleucine, and valine; in some embodiments, X226 is        leucine;    -   L3 is a linker comprising approximately 1-15 amino acids;    -   X228 is an amino acid selected from the group consisting of        arginine, asparagine, aspartic acid, glutamic acid, glutamine,        histidine, lysine, serine, glycine, threonine, and tyrosine; in        some embodiments, X228 is selected from the group consisting of        arginine, asparagine, serine, and threonine; in some        embodiments, X228 is serine.

In some embodiments, each of L1, L2, and L3 has a length and amino acidsequence so that X137, X193, X226, and X228 are arranged with respect toone another in three dimensions space substantially as are residues 137,193, 226, and 228 as shown in FIG. 17 and/or 18, and/or as in an HApolypeptide selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 7. Insome embodiments, L1 comprises approximately 40-70 amino acids; in someembodiments, L1 comprises 50-60 amino acids; in some embodiments, L1comprises 53-58 amino acids. In some embodiments, L1 is approximately 56amino acids long and has an amino acid sequence showing at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% identity with residues 138 to 192 of SEQID NO: 2. In some embodiments, L2 comprises approximately 20-50 aminoacids; in some embodiments, L2 comprises 30-40 amino acids; in someembodiments, L2 comprises 32-35 amino acids. In some embodiments, L2 isapproximately 33 amino acids long and has an amino acid sequencesshowing at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% identity withresidues 194 to 225 of SEQ ID NO: 2. In some embodiments, L3 comprisesapproximately 1-15 amino acids; in some embodiments, L3 comprises 1-10amino acids; in some embodiments, L3 comprises 1-5 amino acids. In someembodiments, L3 is approximately 1 amino acid long and has an amino acidsequences showing 100% identity with residue 227 of SEQ ID NO: 2.

In one aspect, the present invention provides the particular recognitionthat high affinity binding to umbrella-topology glycans alone may not besufficient to confer effective transmission to/infectivity of humans.Rather, the present invention provides the insight that reduced bindingto cone-topology glycans may also be important.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Alignment of exemplary sequences of wild type H2HA. Sequenceswere obtained from the NCBI influenza virus sequence database(www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). Consensus Sequence SEQ IDNO. 1); BAA02771A/Adachi/2/57 (H2N2) (SEQ ID NO. 2); ABP49470A/Albany/1/1958 (H2N2) (SEQ ID NO. 3); ABO44090 A/Albany/1/1959 (H2N2)(SEQ ID NO. 4); ABO01355 A/Albany/1/1960 (H2N2) (SEQ ID NO. 5); ABO52247A/Albany/1/1968 (H2N2) (SEQ ID NO. 6); ACV49600 A/Japan/305/1957 (H2N2)(SEQ ID NO. 7); BAC43764 A/Kayano/57 (H2N2) (SEQ ID NO. 8); ACD88670A/Chicken/PA/298101-4/2004 (H2N2) (SEQ ID NO. 9); ACJ69319A/Chicken/Pennsylvania/SG-00426/2004 (H2N2) (SEQ ID NO. 10); andACJ69324 A/Chicken/Pennsylvania/SG-00426/2004 (H2N2) (SEQ ID NO. 11).

FIG. 2. Alignment of exemplary sequences of wild type H3 Sequences wereobtained from the NCBI influenza virus sequence database(www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). Consensus Sequence (SEQ IDNO. 12); AAB69805 A/Alaska/84/57 (H3N2) (SEQ ID NO. 13); ABP49514A/Albany/10/1968 (H3N2) (SEQ ID NO. 14); ABO58928 A/Bangkok/2/1979(H3N2) (SEQ ID NO. 15); ACF41735 A/Hong Kong/1-1-MA-12/1968 (H3N2) (SEQID NO. 16); ABB54514 A/Memphis/1/1968 (H3N2) (SEQ ID NO. 17); AAT64722A/Netherlands/209/80 (H3N2) (SEQ ID NO. 18); ALL60153 A/Oregon/4/80(H3N2) (SEQ ID NO. 19); and ACH95743 A/Taiwan/VGHYM0109-12/1984 (H3N2)(SEQ ID NO. 20).

FIG. 3. Exemplary cone topologies. This Figure illustrates certainexemplary (but not exhaustive) glycan structures that adopt conetopologies.

FIG. 4. Exemplary umbrella topologies. (A) Certain exemplary (but notexhaustive) N- and O-linked glycan structures that can adopt umbrellatopologies. (B) Certain exemplary (but not exhaustive) O-linked glycanstructures that can adopt umbrella topologies.

FIG. 5. Exemplary glycan receptor-binding specificity of Alb58 HA. (A)Exemplary dose-dependent direct glycan array binding of Alb58 HA whichshows high affinity binding to human receptors in comparison with avianreceptor binding. (B) Exemplary extensive staining of apical surface ofhuman tracheal epithelia and observable staining of alveolar tissuesection by Alb58 HA (lighter) shown against propidium iodide staining(darker).

FIG. 6. Exemplary glycan receptor-binding specificity of mutant forms ofAlb58 HA. Shown in (A-C) are certain exemplary dose-dependent glycanarray binding of Alb58-LG, Alb58-QG and Alb58-QS mutants respectively. Asingle amino acid change from Ser228→Gly (Alb58-LG mutant) leads to aloss of avian receptor binding observed in Alb58 HA. An additionalLeu226→Gln mutation (on Alb58-LG) completely transforms the bindingspecificity by making the Alb58-QG mutant bind predominantly to avianreceptors. Alb58-QS mutant shows loss of both avian and human receptorbinding. (D) Exemplary homology based structural model of Alb58-QSmutant (RBS part is shown as a cartoon) with the human receptor. Boththe Leu226 and Gln226 side chains are marked. The Gln226 in the mutantis positioned to interact with Ser228 hence making the 226 position lessfavorable for contacts with both human and avian receptors.

FIG. 7. Exemplary glycan receptor-binding specificity of CkPA04 HA. (A)Exemplary dose-dependent direct glycan array binding of CkPA04 HA whichshows high affinity binding to avian receptors in comparison with humanreceptors. (B) Exemplary extensive alveolar staining and minimalstaining of apical surface of the human tracheal epithelia by CkPA04 HA(lighter) shown against propidium iodide staining (darker).

FIG. 8. Exemplary homology-based structural model of HA-glycan receptorcomplexes. (A) Exemplary stereo view of the RBS (shown as cartoon) ofCkPA04 HA-avian receptor structural complex constructed using co-crystalstructure of A/Chicken/NY/91-avian receptor (PRB ID: 2WR2) as atemplate. The resolved coordinates of the avian receptor(Neu5Acα2→3Galβ1→3GlcNAc) are shown using a stick representation. (B)Exemplary stereo view of RBS (shown as cartoon) of Alb58 HA—humanreceptor complex constructed using co-crystal structure ofA/Singapore/1/57—human receptor (PDB ID: 2WR7) as the template. Theresolved coordinates of the human receptor(Neu5Aca2→6Galβ1→4GlcNAcβ1→3Gal) are shown using a stick representation.The side chains of the key residues involved in interaction with glycanreceptor are shown and labeled. The residues in the RBS that differbetween CkPA04 and Alb58 HA are underlined.

FIG. 9. Exemplary glycan receptor-binding specificity of mutant forms ofCkPA04 HA. Exemplary dose-dependent glycan receptor binding (A, C, E)and exemplary human tissue binding (B, D, F) of CkPA04-LS, CkPA04-TLSand CkPA04-RTLS mutants respectively. All the mutants show substantialimprovement in the human receptor binding and reduction in avianreceptor binding in comparison to the WT CkPA04 HA as observed in boththe glycan array tissue-binding experiments.

FIG. 10. Exemplary glycan receptor-binding affinities of mutant forms ofCkPA04 HA. (A) Certain exemplary theoretical binding curves (with theapparent binding constant Kd′) that depict the differences in thebinding affinity of the WT and mutant H2N2 HAs to the representativeavian receptor (3′SLN-LN). (B) Certain exemplary theoretical bindingcurves that depict the differences in the binding affinity of the WT andmutant H2N2 HAs to the representative human receptor (6′SLN-LN). Therange of Kd′ values (3-8 pM) is shown for CkPA04-TLS, Alb58 andCkPA04-RTLS that is contrasted with the Kd′ value of CkPA04-LS. Thebinding curves were generated by fitting to the Hill equation (seeMethods) and plotting the theoretically calculated fractional saturation(y-axis) against HA concentration (x-axis). The n value for all thebinding events is around 1.3.

FIG. 11: Conformational map and solvent accessibility of Neu5Acα2-3Galand Neu5Acα2-6Gal motifs. Panel A shows the conformational map ofNeu5Acα2-3Gal linkage. The encircled region 2 is the trans conformationobserved in the APR34_H1_(—)23, ADU63_H3_(—)23 and ADS97_H5_(—)23co-crystal structures. The encircled region 1 is the conformationobserved in the AAI68_H3_(—23) co-crystal structure. Panel B shows theconformational map of Neu5Acα2-6Gal where the cis-conformation(encircled region 3) is observed in all the HA-α2-6 sialylated glycanco-crystal structures. Panel C shows difference between solventaccessible surface area (SASA) of Neu5Ac α2-3 and α2-6 sialylatedoligosaccharides in the respective HA-glycan co-crystal structures. Thebars respectively indicate that Neu5Ac in α2-6 (positive value) or α2-3(negative value) sialylated glycans makes more contact with glycanbinding site. Panel D shows difference between SASA of NeuAc in α2-3sialylated glycans bound to swine and human H1 (H1_(α2-3)), avian andhuman H3 (H3_(α2-3)), and of NeuAc in α2-6 sialylated glycans bound toswine and human H1 (H1_(α2-6)). The negative bar for H3_(α2-3) indicateslesser contact of the human H3 HA with Neu5Acα2-3Gal compared to that ofavian H3. Torsion angles—φ: C2-C1-O—C3 (for Neu5Acα2-3/6 linkage); ψ:C1-O—C3-H3 (for Neu5Acα2-3Gal) or C1-O—C6-C5 (for Neu5Acα2-6Gal); ω:O—C6-C5-H5 (for Neu5Acα2-6Gal) linkages.

The φ, ψ maps were obtained from GlycoMaps DB(http://www.glycosciences.de/modeling/glycomapsdb/) which was developedby Dr. Martin Frank and Dr. Claus-Wilhelm von der Lieth (German CancerResearch Institute, Heidelberg, Germany). The coloring scheme from highenergy to low energy is from darker to lighter, respectively.

FIG. 12: Framework for understanding glycan receptor specificity. α2-3-and/or α2-6-linked glycans can adopt different topologies. In someembodiments, the ability of an HA polypeptide to bind to certain ofthese topologies confers upon it the ability to mediate infection ofdifferent hosts, for example, humans. As illustrated in Panel A of thisfigure, there are two particularly relevant topologies, a “cone”topology and an “umbrella” topology. The cone topology can be adopted byα2-3- and/or α2-6-linked glycans, and is typical of shortoligosaccharides or branched oligosaccharides attached to a core(although this topology can be adopted by certain longoligosaccharides). The umbrella topology can only be adopted byα2-6-linked glycans (presumably due to the increased conformationalplurality afforded by the extra C5-C6 bond that is present in the α2-6linkage), and is predominantly adopted by long oligosaccharides orbranched glycans with long oligosaccharide branches, particularlycontaining the motif Neu5Acα2-6Galβ1-3/4GlcNAc-. As described herein,ability of HA polypeptides to bind the umbrella glycan topology, confersbinding to human receptors and/or ability to mediate infection ofhumans. Panel B of this Figure specifically shows the topology of α2-3and α2-6 as governed by the glycosidic torsion angles of thetrisaccharide motifs—Neu5Acα2-3Galβ1-3/4GlcNAc andNeu5Acα2-6Galβ1-4GlcNAc respectively. A parameter (θ)—angle between C2atom of Neu5Ac and C1 atoms of the subsequent Gal and GlcNAc sugars inthese trisaccharide motifs was defined to characterize the topology.Superimposition of the θ contour and the conformational maps of the α2-3and α2-6 motifs shows that α2-3 motifs adopt 100% cone-like topology andα2-6 motifs sampled both cone-like and umbrella-like topologies (PanelC). In the cone-like topology sampled by α2-3 and α2-6, GlcNAc andsubsequent sugars are positioned along a region spanning a cone.Interactions of HA with cone-like topology primarily involve contacts ofamino acids at the numbered positions (based on H3 HA numbering) withNeu5Ac and Gal sugars. On the other hand, in umbrella-like topology,which is unique to α2-6, \GlcNAc and subsequent sugars bend towards theHA binding site (as observed in HA-α2-6 co-crystal structures). Longerα2-6 oligosaccharides (e.g. at least a tetrasaccharide) would favor thisconformation since it is stabilized by intra-sugar van der Waals contactbetween acetyl groups of GlcNAc and Neu5Ac. HA interactions withumbrella-like topology involve contacts of amino acids at the numberedpositions (based on H3 HA numbering) with GlcNAc and subsequent sugarsin addition to contacts with Neu5Ac and Gal sugars. Panel C of thisFigure depicts conformational sampling of cone- and umbrella-liketopology by α2-3 and α2-6. Sections (A)-(D) show the conformational (φ,ψ) maps of Neu5Acα2-3Gal, Neu5Acα2-6Gal, Galβ1-3GlcNAc, andGalβ1-4GlcNAc linkages, respectively. These maps obtained from GlycoMapsDB (http://www.glycosciences.de/modeling/glycomapsdb/) were generatedusing ab initio MD simulations using MM3 force field. Energydistribution is color coded starting from darker (representing higherenergy) to lighter representing lower energy. Encircled regions 1-5represent (φ,ψ), values observed for the α2-3 and α2-6 oligosaccharidesin the HA-glycan co-crystal structures. The trans conformation(encircled region 1) of Neu5Acα2-3Gal predominates in HA binding pocketwith the exception of the co-crystal structure of A/Aichi/2/68 H3N2 HAwith α2-3 where this conformation is gauche (encircled region 2). On theother hand, the cis conformation of Neu5Acα2-6Gal (encircled region 3)predominates in HA binding pocket. The cone-like topology is sampled byencircled regions 1 and 2 and the umbrella-like topology is sampled byencircled region 3. Sections (E)-(F) show sampling of cone-like andumbrella-like topologies by α2-3 and α2-6 motifs, respectively. Thedarker regions in the conformational maps were used as the outerboundaries to calculate the θ parameter (angle between C2 atom of Neu5Acand C1 atoms of subsequent Gal and GlcNAc sugars) for a given set of(φ,ψ), values. Based on the energy cutoff, the value of θ>110° was usedto characterize cone-like topology and θ<100° was used to characterizeumbrella-like topology. Superimposition of the θ contour with theconformational energy map indicated that α2-3 motif adopts 100%cone-like topology since it was energetically unfavorable to adoptumbrella-like topology. On the other hand, the α2-6 motif sampled boththe cone-like and umbrella-like topologies and this sampling wasclassified based on the ω angle (O-C6-C5-H5) of Neu5Acα2-6Gal linkage.

FIG. 13. Interactions of HA residues with cone vs umbrella glycantopologies. Analysis of HA-glycan co-crystals reveals that the positionof Neu5Ac relative to the HA binding site is almost invariant. Contactswith Neu5Ac involve highly conserved residues such as F98, S/T136, W153,H183 and L/1194. Contacts with other sugars involve different residues,depending on whether the sugar linkage is α2-3 or α2-6 and whether theglycan topology is cone or umbrella. For example, in the cone topology,the primary contacts are with Neu5Ac and with Gal sugars. E190 and Q226play particularly important roles in this binding. This Figure alsoillustrates other positions (e.g., 137, 145, 186, 187, 193, 222) thatcan participate in binding to cone structures. In some cases, differentresidues can make different contacts with different glycan structures.The type of amino acid in these positions can influence ability of an HApolypeptide to bind to receptors with different modification and/orbranching patterns in the glycan structures. In the umbrella topology,contacts are made with sugars beyond Neu5Ac and Gal. This Figureillustrates residues (e.g., 137, 145, 156, 159, 186, 187, 189, 190, 192,193, 196, 222, 225, 226) that can participate in binding to umbrellastructures. In some cases, different residues can make differentcontacts with different glycan structures. The type of amino acid inthese positions can influence ability of an HA polypeptide to bind toreceptors with different modification and/or branching patterns in theglycan structures. In some embodiments, a D residue at position 190and/or a D residue at position 225 contribute(s) to binding to umbrellatopologies.

FIG. 14. Glycan profile of human bronchial epithelial cells and humancolonic epithelial cells. To further investigate the glycan diversity inthe upper respiratory tissues, N-linked glycans were isolated from HBEs(a representative upper respiratory cell line) and analyzed usingMALDI-MS. The predominant expression of a2-6 in HBEs was confirmed bypre-treating the sample with Sialidase S (a2-3 specific) and Sialidase A(cleaves and SA). The predominant expression of glycans with long branchtopology is supported by TOF-TOF fragmentation analysis ofrepresentative mass peaks. To provide a reference for glycan diversityin the upper respiratory tissues, the N-linked glycan profile of humancolonic epithelial cells (HT29; a representative gut cell line) wasobtained. This cell line was chosen because the current H5N1 viruseshave been shown to infect gut cells. Sialidase A and S pre-treatmentcontrols showed predominant expression of a2-3 glycans in the HT-29cells. Moreover, the long branch glycan topology is not as prevalent asobserved for HBEs. Therefore, human adaptation of the H5N1 HA wouldinvolve HA mutations that would enable high affinity binding to thediverse glycans expressed in the human upper respiratory tissues (e.g.,umbrella glycans).

FIG. 15. Data mining platform. Shown in (A) are the main components ofthe data mining platform. The features are derived from the data objectswhich are extracted from the database. The features are prepared intodatasets that are used by the classification methods to derive patternsor rules (B), shows the key software modules that enable the user toapply the data mining process to the glycan array data.

FIG. 16. Features used in data mining analysis. This figure shows thefeatures defined herein as representative motifs that illustrate thedifferent types of pairs, triplets and quadruplets abstracted from theglycans on the glycan microarray. The rationale behind choosing thesefeatures is based on the binding of di-tetra saccharides to the glycanbinding site of HA. The final dataset comprise features from the glycansas well as the binding signals for each of the HAs screened on thearray. Among the different methods for classification, the ruleinduction classification method was utilized. One of the main advantagesof this method is that it generates IF-THEN rules which can beinterpreted more easily when compared to the other statistical ormathematical methods. The two main objectives of the classificationwere: (1) identifying features present on a set of high affinity glycanligands, which enhance binding, and (2) identifying features that are inthe low affinity glycan ligands that are not favorable for binding.

FIG. 17. Crystal Structure of Exemplary H2 HA. (a) Chemical structuresof α2,3- and α2,6-linked glycans, with the terminal sialic acid andgalactose shown here. (b) Overview of the 1957 H2 trimer. Five potentialglycosylation sites are found on each monomer (as labeled). Glycans inthe density map are shown. (c) Receptor binding site of H2. Residuesinvolved in receptor binding, as suggested by the H3 structures, areshown in sticks. Aromatic residues comprising the base of the bindingsite are absolutely conserved in various HA subtypes. Residues from the220 loop and position 190 are critical for the receptor specificityswitch in H1, H2, and H3. (Xu R et al., J Virol 84(4):1715-1721, 2010).

FIG. 18. Interactions of avian H2 HA and human H2 HA with avian andhuman receptor analogs. Interactions of an avian H2 HA (upper panels)and a human H2 HA (lower panels) with avian and human receptoranalogues. The three secondary structure elements of the binding site,the 130- and 220-loops and the 190-helix are labeled in this backbonerepresentation together with some selected side chains in stickrepresentation. The broken lines indicate potential hydrogen bondinteraction. In all four panels, the carbon, nitrogen, and oxygen atomsin the sialosaccharides are depicted, and water molecules are labeled.A/dk/Ontario/77 H2 HA in complex with avian receptor, LSTa, (A) andhuman receptor, LSTc. (B). ASingapore/1/57 H2 HA in complex with humanreceptor (C) and avian receptor (D). The black arrows in A, B, and Cindicate that for the two human receptor complexes the Sia-1/Gal-2linkage adopts a cis conformation, whereas for the avian complex itadopts a trans conformation (Liu J, et al. 2009 Proc Natl Acad Sci USA106(40); 17175-17180; incorporated herein by reference).

DESCRIPTION OF HA SEQUENCE ELEMENTS

HA Sequence Element 1

HA Sequence Element 1 is a sequence element corresponding approximatelyto residues 97-185 (where residue positions are assigned using H3 HA asreference) of many HA proteins found in natural influenza isolates. Thissequence element has the basic structure:

(SEQ ID NO. 21) C (Y/F) P X₁ C X₂ W X₃ W X₄ H H P, wherein:

-   -   X₁ is approximately 30-45 amino acids long;    -   X₂ is approximately 5-20 amino acids long;    -   X₃ is approximately 25-30 amino acids long; and    -   X₄ is approximately 2 amino acids long.

In some embodiments, X₁ is about 35-45, or about 35-43, or about 35, 36,37, 38, 38, 40, 41, 42, or 43 amino acids long. In some embodiments, X₂is about 9-15, or about 9-14, or about 9, 10, 11, 12, 13, or 14 aminoacids long. In some embodiments, X₃ is about 26-28, or about 26, 27, or28 amino acids long. In some embodiments, X₄ has the sequence (G/A)(I/V). In some embodiments, X₄ has the sequence GI; in some embodiments,X₄ has the sequence GV; in some embodiments, X₄ has the sequence AI; insome embodiments, X₄ has the sequence AV. In some embodiments, HASequence Element 1 comprises a disulfide bond. In some embodiments, thisdisulfide bond bridges residues corresponding to positions 97 and 139(based on the canonical H3 numbering system utilized herein).

HA Sequence Element 2

HA Sequence Element 2 is a sequence element corresponding approximatelyto residues 324-340 (again using a numbering system based on H3 HA) ofmany HA proteins found in natural influenza isolates. This sequenceelement has the basic structure:

-   -   G A I A G F I E (SEQ ID NO. 22)        In some embodiments, HA Sequence Element 2 has the sequence:    -   P X₁G A I A G F I E, (SEQ ID NO. 23) wherein:    -   X₁ is approximately 4-14 amino acids long, or about 8-12 amino        acids long, or about 12, 11, 10, 9 or 8 amino acids long. In        some embodiments, this sequence element provides the HA0        cleavage site, allowing production of HA1 and HA2.        HA Sequence Element 2

HA Sequence Element 2 is a sequence element corresponding approximatelyto residues 324-340 (again using a numbering system based on H3 HA) ofmany HA proteins found in natural influenza isolates. This sequenceelement has the basic structure:

GAIAGFIEIn some embodiments, HA Sequence Element 2 has the sequence:

PX₁GAIAGFIE, wherein:

-   -   X₁ is approximately 4-14 amino acids long, or about 8-12 amino        acids long, or about 12, 11, 10, 9 or 8 amino acids long. In        some embodiments, this sequence element provides the HA0        cleavage site, allowing production of HA1 and HA2.

DEFINITIONS

Affinity: As is known in the art, “affinity” is a measure of thetightness with a particular ligand (e.g., an HA polypeptide) binds toits partner (e.g., an HA receptor). Affinities can be measured indifferent ways. In some embodiments, affinity is measured by aquantitative assay (e.g., glycan binding assays). In some suchembodiments, binding partner concentration (e.g., HA receptor, glycan,etc.) may be fixed to be in excess of ligand (e.g., an HA polypeptide)concentration so as to mimic physiological conditions (e.g., viral HAbinding to cell surface glycans). Alternatively or additionally, in someembodiments, binding partner (e.g., HA receptor, glycan, etc.)concentration and/or ligand (e.g., an HA polypeptide) concentration maybe varied. In some such embodiments, affinity (e.g., binding affinity)may be compared to a reference (e.g., a wild type HA that mediatesinfection of a humans) under comparable conditions (e.g.,concentrations).

Binding: It will be understood that the term “binding”, as used herein,typically refers to a non-covalent association between or among agents.In many embodiments herein, binding is addressed with respect toparticular glycans (e.g., umbrella topology glycans or cone topologyglycans). It will be appreciated by those of ordinary skill in the artthat such binding may be assessed in any of a variety of contexts. Insome embodiments, binding is assessed with respect to free glycans. Insome embodiments, binding is assessed with respect to glycans attached(e.g., covalently linked to) a carrier. In some such embodiments, thecarrier is a polypeptide. In some embodiments, binding is assessed withrespect to glycans attached to an HA receptor. In such embodiments,reference may be made to receptor binding or to glycan binding.

Binding agent: In general, the term “binding agent” is used herein torefer to any entity that binds to glycans (e.g., to umbrella-topologyglycans) as described herein. Binding agents may be of any chemicaltype. In some embodiments, binding agents are polypeptides (including,e.g., antibodies or antibody fragments); in some such embodiments,binding agents are HA polypeptides; in other embodiments, binding agentsare polypeptides whose amino acid sequence does not include an HAcharacteristic sequence (i.e., “Non-HA polypeptides). In someembodiments, binding agents are small molecules. In some embodiments,binding agents are nucleic acids. In some embodiments, binding agentsare aptamers. In some embodiments, binding agents are polymers; in someembodiments, binding agents are non-polymeric. In some embodiments,binding agents are carbohydrates. In some embodiments, binding agentsare lectins. In some embodiments, binding agents as described hereinbind to sialylated glycans having an umbrella-like topology. In someembodiments, binding agents bind to umbrella-topology glycans with highaffinity and/or specificity. In some embodiments, binding agents show abinding preference for umbrella-topology glycans as compared withcone-topology glycans. In some embodiments, binding agents compete withhemagglutinin for binding to glycans on hemagglutinin receptors. In someembodiments, binding agents compete with hemagglutinin for binding toumbrella-topology glycans. In some embodiments, a binding agent providedherein is an umbrella topology blocking agent. In some embodiments, abinding agent provided herein is an umbrella topology specific blockingagent. In some embodiments, binding agents bind to umbrella topologyglycan mimics.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active. In particularembodiments, where a protein or polypeptide is biologically active, aportion of that protein or polypeptide that shares at least onebiological activity of the protein or polypeptide is typically referredto as a “biologically active” portion.

Characteristic portion: As used herein, the phrase a “characteristicportion” of a protein or polypeptide is one that contains a continuousstretch of amino acids, or a collection of continuous stretches of aminoacids, that together are characteristic of a protein or polypeptide.Each such continuous stretch generally will contain at least two aminoacids. Furthermore, those of ordinary skill in the art will appreciatethat typically at least 5, at least 10, at least 15, at least 20 or moreamino acids are required to be characteristic of a protein. In general,a characteristic portion is one that, in addition to the sequenceidentity specified above, shares at least one functional characteristicwith the relevant intact protein.

Characteristic sequence: A “characteristic sequence” is a sequence thatis found in all members of a family of polypeptides or nucleic acidsand/or that includes an immunogenic epitope, and therefore can be usedby those of ordinary skill in the art to define members of the family.

Cone topology: The phrase “cone topology” is used herein to refer to a3-dimensional arrangement adopted by certain glycans and in particularby glycans on HA receptors. As illustrated in FIG. 3, the cone topologycan be adopted by α2-3 sialylated glycans or by α2-6 sialylated glycans,and is typical of short oligonucleotide chains, though some longoligonucleotides can also adopt this conformation. The cone topology ischaracterized by the glycosidic torsion angles of Neu5Acα2-3Gal linkagewhich samples three regions of minimum energy conformations given by φ(C1-C2-O—C3/C6) value of about −60, about 60 or about 180 and ψ(C2-O—C3/C6-H3/C5) samples −60 to 60 (FIG. 11). FIG. 3 presents certainrepresentative (though not exhaustive) examples of glycans that adopt acone topology.

Corresponding to: As used herein, the term “corresponding to” is oftenused to designate the position/identity of an amino acid residue in anHA polypeptide. Those of ordinary skill will appreciate that, forpurposes of simplicity, a canonical numbering system (based on wild typeH3 HA) is utilized herein (as illustrated, for example, in FIGS. 1-2),so that an amino acid “corresponding to” a residue at position 190, forexample, need not actually be the 190^(th) amino acid in a particularamino acid chain but rather corresponds to the residue found at 190 inwild type H3 HA; those of ordinary skill in the art readily appreciatehow to identify corresponding amino acids.

Degree of separation removed: As used herein, amino acids that are a“degree of separation removed” are HA amino acids that have indirecteffects on glycan binding. For example, one-degree-of-separation-removedamino acids may either: (1) interact with the direct-binding aminoacids; and/or (2) otherwise affect the ability of direct-binding aminoacids to interact with glycan that is associated with host cell HAreceptors; such one-degree-of-separation-removed amino acids may or maynot directly bind to glycan themselves. Two-degree-of-separation-removedamino acids either (1) interact with one-degree-of-separation-removedamino acids; and/or (2) otherwise affect the ability of theone-degree-of-separation-removed amino acids to interact withdirect-binding amino acids, etc.

Direct-binding amino acids: As used herein, the phrase “direct-bindingamino acids” refers to HA polypeptide amino acids which interactdirectly with one or more glycans that is associated with host cell HAreceptors.

Engineered: The term “engineered”, as used herein, describes apolypeptide whose amino acid sequence has been selected by man. Forexample, an engineered HA polypeptide has an amino acid sequence thatdiffers from the amino acid sequences of HA polypeptides found innatural influenza isolates. In some embodiments, an engineered HApolypeptide has an amino acid sequence that differs from the amino acidsequence of HA polypeptides included in the NCBI database.

H2 polypeptide: An “H2 polypeptide”, as that term is used herein, is anHA polypeptide whose amino acid sequence includes at least one sequenceelement that is characteristic of H2 and distinguishes H2 from other HAsubtypes. Representative such sequence elements can be determined byalignments such as, for example, those illustrated in FIG. 1 andinclude, for example, those described herein with regard to H2-specificembodiments of HA Sequence Elements.

Hemagglutinin (HA) polypeptide: As used herein, the term “hemagglutininpolypeptide” (or “HA polypeptide”) refers to a polypeptide whose aminoacid sequence includes at least one characteristic sequence of HA. Awide variety of HA sequences from influenza isolates are known in theart; indeed, the National Center for Biotechnology Information (NCBI)maintains a database (www.ncbi.nlm.nih.gov/genomes/FLU/flu.html) that,as of the filing of the present application included 9796 HA sequences.Those of ordinary skill in the art, referring to this database, canreadily identify sequences that are characteristic of HA polypeptidesgenerally, and/or of particular HA polypeptides (e.g., H1, H2, H3, H4,H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16 polypeptides;or of HAs that mediate infection of particular hosts, e.g., avian,camel, canine, cat, civet, environment, equine, human, leopard, mink,mouse, seal, stone martin, swine, tiger, whale, etc. For example, insome embodiments, an HA polypeptide includes one or more characteristicsequence elements found between about residues 97 and about 185, about324 and about 340, about 96 and about 100, and/or about 130 and about230 of an HA protein found in a natural isolate of an influenza virus.In some embodiments, an HA polypeptide has an amino acid sequencecomprising at least one of HA Sequence Elements 1 and 2, as definedherein. In some embodiments, an HA polypeptide has an amino acidsequence comprising HA Sequence Elements 1 and 2, in some embodimentsseparated from one another by about 100 to about 200, or by about 125 toabout 175, or about 125 to about 160, or about 125 to about 150, orabout 129 to about 139, or about 129, about 130, about 131, about 132,about 133, about 134, about 135, about 136, about 137, about 138, orabout 139 amino acids. In some embodiments, an HA polypeptide has anamino acid sequence that includes residues at positions within theregions 96-100 and/or 130-230 that participate in glycan binding. Forexample, many HA polypeptides include one or more of the followingresidues: Tyr98, Ser/Thr136, Trp153, H is 183, and Leu/Ile194. In someembodiments, an HA polypeptide includes at least 2, 3, 4, or all 5 ofthese residues.

High affinity binding: The term “high affinity binding”, as used hereinrefers to a high degree of tightness with which a particular ligand(e.g., an HA polypeptide) binds to its partner (e.g., an HA receptor).Affinities can be measured by any available method, including thoseknown in the art. In some embodiments, binding is considered to be highaffinity if the Kd′ is about 500 pM or less (e.g., below about 400 pM,about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM,about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about20 pM, about 10 pM, about 5 pM, about 4 pM, about 3 pM, about 2 pM,etc.) in binding assays. In some embodiments, binding is considered tobe high affinity if the affinity is stronger (e.g., the Kd′ is lower)for a polypeptide of interest than for a selected reference polypeptide.In some embodiments, binding is considered to be high affinity if theratio of the Kd′ for a polypeptide of interest to the Kd′ for a selectedreference polypeptide is 1:1 or less (e.g., 0.9:1, 0.8:1, 0.7:1, 0.6:1,0.5:1, 0.4:1, 0.3:1, 0.2:1, 0.1:1, 0.05:1, 0.01:1, or less). In someembodiments, binding is considered to be high affinity if the Kd′ for apolypeptide of interest is about 100% or less (e.g., about 99%, about98%, about 97%, about 96%, about 95%, about 90%, about 85%, about 80%,about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%,about 10%, about 5%, about 4%, about 3%, about 2%, about 1% or less) ofthe Kd′ for a selected reference polypeptide.

Isolated: The term “isolated”, as used herein, refers to an agent orentity that has either (i) been separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature or in an experimental setting); or (ii) produced by the handof man. Isolated agents or entities may be separated from at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure.

Linkage Specific Blocking Agent (LSBA): As used herein, the term“linkage specific blocking agent” refers to an agent which binds to anHA receptor having an α2-6 sialylated glycan. In some embodiments, anLSBA selectively binds to an HA receptor having an α2-6 sialylatedglycan with at least about 40, 50, or 75% of the affinity of that for anHA receptor having an α2-3 sialylated glycan. In some embodiments, anLSBA selectively binds to an HA receptor having an α2-6 sialylatedglycan with at least about 2, 4, 5, or 10 times greater affinity thanthat for an HA receptor having an α2-3 sialylated glycan. In someembodiments, an LSBA has an affinity for an α2-6 sialylated glycan thatis at least 50, 100, 150, or 200% of its affinity for an α2-3 sialylatedglycan. In some embodiments, an LSBA may compete with hemagglutinin forbinding to an HA receptor. For example, an LSBA may selectively inhibitthe binding of an influenza virus particle (e.g., human or avianinfluenza virus) to an HA receptor based on the linkage characteristics(e.g., α2-6 sialylated glycan or α2-3 sialylated glycan). In someembodiments, an LSBA is a polypeptide. In some such embodiments, an LSBApolypeptide has an amino acid sequence that is substantially identicalor substantially homologous to that of a naturally-occurringpolypeptide. In some embodiments, an LSBA polypeptide is an HApolypeptide. In some embodiments, an LSBA polypeptide is anaturally-occurring HA polypeptide, or a fragment thereof. In someembodiments, an LSBA polypeptide has an amino acid sequence that is notrelated to that of an HA polypeptide. In some embodiments, an LSBApolypeptide is an antibody or fragment thereof. In some embodiments, anLSBA polypeptide is a lectin (e.g., SNA-1). In some embodiments, an LSBAis not a polypeptide. In some embodiments, an LSBA is a small molecule.In some embodiments, an LSBA is a nucleic acid.

Long oligosaccharide: For purposes of the present disclosure, anoligosaccharide is typically considered to be “long” if it includes atleast one linear chain that has at least four saccharide residues.

Low affinity binding: The term “low affinity binding”, as used hereinrefers to a low degree of tightness with which a particular ligand(e.g., an HA polypeptide) binds to its partner (e.g., an HA receptor).As described herein, affinities can be measured by any available method,including methods known in the art. In some embodiments, binding isconsidered to be low affinity if the Kd′ is about 100 pM or more (e.g.,above about 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900pM, 1 nM, 1.1 nM, 1.2 nM, 1.3 nM, 1.4 nM, 1.5 nM, etc.) In someembodiments, binding is considered to be low affinity if the affinity isthe same or lower (e.g., the Kd′ is about the same or higher) for apolypeptide of interest than for a selected reference polypeptide. Insome embodiments, binding is considered to be low affinity if the ratioof the Kd′ for a polypeptide of interest to the Kd′ for a selectedreference polypeptide is 1:1 or more (e.g., 1.1:1, 1.2:1, 1.3:1, 1.4:1,1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 3:1, 4:1, 5:1, 10:1 or more). Insome embodiments, binding is considered to be low affinity if the Kd′for a polypeptide of interest is 100% or more (e.g., 100%, 105%, 110%,115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%,175%, 180%, 185%, 190%, 195%, 200%, 300%, 400%, 500%, 1000%, or more) ofthe Kd′ for a selected reference polypeptide.

Non-natural amino acid: The phrase “non-natural amino acid” refers to anentity having the chemical structure of an amino acid (i.e.:

and therefore being capable of participating in at least two peptidebonds, but having an R group that differs from those found in nature. Insome embodiments, non-natural amino acids may also have a second R grouprather than a hydrogen, and/or may have one or more other substitutionson the amino or carboxylic acid moieties.

Polypeptide: A “polypeptide”, generally speaking, is a string of atleast two amino acids attached to one another by a peptide bond. In someembodiments, a polypeptide may include at least 3-5 amino acids, each ofwhich is attached to others by way of at least one peptide bond. Thoseof ordinary skill in the art will appreciate that polypeptides sometimesinclude “non-natural” amino acids or other entities that nonetheless arecapable of integrating into a polypeptide chain, optionally.

Predominantly Present: The term “predominantly present”, as used herein,refers to the presence of an entity (e.g., an amino acid residue) at aparticular location across a population. For example, an amino acid maybe predominantly present if, across a population of polypeptides, aparticular amino acid is statistically present in at least about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% ormore of the population of polypeptides.

Prevention: The term “prevention”, as used herein, refers to a delay ofonset, and/or reduction in frequency and/or severity of one or moresymptoms of a particular disease, disorder or condition (e.g., infectionfor example with influenza virus). In some embodiments, prevention isassessed on a population basis such that an agent is considered to“prevent” a particular disease, disorder or condition if a statisticallysignificant decrease in the development, frequency, and/or intensity ofone or more symptoms of the disease, disorder or condition is observedin a population susceptible to the disease, disorder, or condition.

Pure: As used herein, an agent or entity is “pure” if it issubstantially free of other components. For example, a preparation thatcontains more than about 90% of a particular agent or entity istypically considered to be a pure preparation. In some embodiments, anagent or entity is at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% pure.

Short oligosaccharide: For purposes of the present disclosure, anoligosaccharide is typically considered to be “short” if it has fewerthan 4, or certainly fewer than 3, residues in any linear chain.

Specificity: As is known in the art, “specificity” is a measure of theability of a particular ligand (e.g., an HA polypeptide) to distinguishits binding partner (e.g., a human HA receptor, and particularly a humanupper respiratory tract HA receptor) from other potential bindingpartners (e.g., an avian HA receptor).

Substantial homology: The phrase “substantial homology” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially homologous” ifthey contain homologous residues in corresponding positions. Homologousresidues may be identical residues. Alternatively, homologous residuesmay be non-identical residues will appropriately similar structuraland/or functional characteristics. For example, as is well known bythose of ordinary skill in the art, certain amino acids are typicallyclassified as “hydrophobic” or “hydrophilic” amino acids, and/or ashaving “polar” or “non-polar” side chains Substitution of one amino acidfor another of the same type may often be considered a “homologous”substitution. Typical amino acid categorizations are summarized below:

Alanine Ala A nonpolar neutral 1.8 Arginine Arg R polar positive −4.5Asparagine Asn N polar neutral −3.5 Aspartic Asp D polar negative −3.5acid Cysteine Cys C nonpolar neutral 2.5 Glutamic Glu E polar negative−3.5 acid Glutamine Gln Q polar neutral −3.5 Glycine Gly G nonpolarneutral −0.4 Histidine His H polar positive −3.2 Isoleucine Ile Inonpolar neutral 4.5 Leucine Leu L nonpolar neutral 3.8 Lysine Lys Kpolar positive −3.9 Methionine Met M nonpolar neutral 1.9 PhenylalaninePhe F nonpolar neutral 2.8 Proline Pro P nonpolar neutral −1.6 SerineSer S polar neutral −0.8 Threonine Thr T polar neutral −0.7 TryptophanTrp W nonpolar neutral −0.9 Tyrosine Tyr Y polar neutral −1.3 Valine ValV nonpolar neutral 4.2

Ambiguous Amino Acids 3-Letter 1-Letter Asparagine or aspartic acid AsxB Glutamine or glutamic acid Glx Z Leucine or Isoleucine Xle JUnspecified or unknown amino acid Xaa XAs is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999;all of the foregoing of which are incorporated herein by reference. Inaddition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, at least 55%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or more of their correspondingresidues are homologous over a relevant stretch of residues. In someembodiments, the relevant stretch is a complete sequence. In someembodiments, the relevant stretch is at least 10, at least 15, at least20, at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 95, at least 100, atleast 125, at least 150, at least 175, at least 200, at least 225, atleast 250, at least 275, at least 300, at least 325, at least 350, atleast 375, at least 400, at least 425, at least 450, at least 475, atleast 500 or more residues.

Substantial identity: The phrase “substantial identity” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially identical” ifthey contain identical residues in corresponding positions. As is wellknown in this art, amino acid or nucleic acid sequences may be comparedusing any of a variety of algorithms, including those available incommercial computer programs such as BLASTN for nucleotide sequences andBLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplarysuch programs are described in Altschul, et al., Basic local alignmentsearch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al.,Methods in Enzymology; Altschul, et al., “Gapped BLAST and PSI-BLAST: anew generation of protein database search programs”, Nucleic Acids Res.25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical Guideto the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al.,(eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132), Humana Press, 1999; all of the foregoing of whichare incorporated herein by reference. In addition to identifyingidentical sequences, the programs mentioned above typically provide anindication of the degree of identity. In some embodiments, two sequencesare considered to be substantially identical if at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or more of their corresponding residues are identicalover a relevant stretch of residues. In some embodiments, the relevantstretch is a complete sequence. In some embodiments, the relevantstretch is at least 10, at least 15, at least 20, at least 25, at least30, at least 35, at least 40, at least 45, at least 50, at least 55, atleast 60, at least 65, at least 70, at least 75, at least 80, at least85, at least 90, at least 95, at least 100, at least 125, at least 150,at least 175, at least 200, at least 225, at least 250, at least 275, atleast 300, at least 325, at least 350, at least 375, at least 400, atleast 425, at least 450, at least 475, at least 500 or more residues.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that elicits a desired biological or pharmacologicaleffect.

Treatment: As used herein, the term “treatment” refers to any methodused to alleviate, delay onset, reduce severity or incidence, or yieldprophylaxis of one or more symptoms or aspects of a disease, disorder,or condition. For the purposes of the present invention, treatment canbe administered before, during, and/or after the onset of symptoms.

Umbrella topology: The phrase “umbrella topology” is used herein torefer to a 3-dimensional arrangement adopted by certain glycans and inparticular by glycans on HA receptors. The present invention encompassesthe recognition that binding to umbrella topology glycans ischaracteristic of HA proteins that mediate infection of human hosts. Asillustrated in FIG. 12, the umbrella topology is typically adopted onlyby α2-6 sialylated glycans, and is typical of long (e.g., greater thantetrasaccharide) oligosaccharides. In some embodiments,umbrella-topology glycans are glycans exhibiting a three-dimensionalstructure substantially similar to the structure presented in FIG. 6(right panel). In some embodiments, umbrella-topology glycans areglycans which contact HA polypeptides via the amino acid residues shownin FIG. 6 (right panel). In some embodiments, umbrella-topology glycansare glycans which are able to contact and/or specifically bind to theamino acid binding pocket shown in FIG. 6 (right panel). In someembodiments, glycan structural topology is classified based on parameterθ defined as angle between C₂ of Sia, C₁ of Gal, and C₁ of GlcNAc.Values of θ<100° represent cone-like topology adopted by α2-3 and shortα2-6 glycans. Values of θ>110° represent umbrella-like topology, such astopology adopted by long α2-6 glycans (FIG. 6). An example of umbrellatopology is given by φ angle of Neu5Acα2-6Gal linkage of around −60(see, for example, FIG. 11). FIG. 4 presents certain representative(though not exhaustive) examples of glycans that can adopt an umbrellatopology. The long α2-6 motifs presented in FIG. 4 includes Neu5Acα2-6linked at the non-reducing end to a long chain (e.g., at least atrisaccharide) found as a part of biological N-linked glycans, O-linkedglycans, and glycolipids. The boxed inset shows examples of theumbrella-topology long α2-6 glycan moieties that are found as a part ofbiological glycans that bind to high affinity with HA. In someembodiments, umbrella-topology glycans (e.g., at a site) comprise agreater proportion of long (e.g. multiple lactosamine units) α2-6oligosaccharide branches than short α2-6 (e.g. single lactosamine)branches. In some embodiments, umbrella-topology glycans (e.g., at asite) comprise about 2-fold, about 3-fold, about 4-fold, about 5-fold,about 10-fold, about 20-fold, about 50-fold, or greater than about50-fold more long α2-6 oligosaccharide branches than short α2-6 (e.g.single lactosamine) branches. In some embodiments, the uniquecharacteristic of HA interactions with umbrella-topology glycans and/orglycan decoys is the HA contact with a glycan comprising sialic acid(SA) and/or SA analogs at the non-reducing end. In some embodiments,chain length of the oligosaccharide is at least a trisaccharide(excluding the SA or SA analog). In some embodiments, a combination ofthe numbered residues shown in the right-hand panel of FIG. 12 isinvolved in contacts with umbrella-like topology. In some embodiments,umbrella topology glycans are oligosaccharides of the following form:

Neu5Acα2-6Sug1-Sug2-Sug3

where:

(a) Neu5Ac α2-6 is typically (but not essentially) at the non-reducingend;

(b) Sug1:

-   -   (i) is a hexose (frequently Gal or Glc) or hexosamine (GlcNAc or        GalNAc) in α or β configuration (frequently β- for N- and        O-linked extension and α- in the case of GalNAcα- that is        O-linked to glycoprotein);    -   (ii) no sugars other than Neu5Acα2-6 are attached to any of the        non-reducing positions of Sug1 (except when Sug1 is GalNAcα-        that is O-linked to the glycoprotein); and/or    -   (iii) non-sugar moieties such as sulfate, phosphate, guanidium,        amine, N-acetyl, etc. can be attached to non-reducing positions        (typically 6 position) of Sug1 (e.g., to improve contacts with        HA);

(c) Sug2 and/or Sug3 is/are:

-   -   (i) hexose (frequently Gal or Glc) or hexosamine (GlcNAc or        GalNAc) in α or β configuration (frequently β); and/or    -   (ii) sugars (such as Fuc) or non-sugar moieties such as sulfate,        phosphate, guanidium, amine, N-acetyl, etc. can be attached to        non-reducing positions of Sug2, Sug3, and/or Sug4;

(d) Linkage between any two sugars in the oligosaccharide apart fromNeu5Acα2-6 linkage can be 1-2, 1-3, 1-4, and/or 1-6 (typically 1-3 or1-4); and/or

(e) Structure where Neu5Acα2-6 is linked GalNAcα that is O-linked to theglycoprotein and additional sugars are linked to the non-reducing end ofGalNAcα for example

(i) Neu5Acα2-6(Neu5Acα2-3Galβ1-3)GalNAcα- (ii)Neu5Acα2-6(Galβ1-3)GalNAcα-

Umbrella topology blocking agent (UTBA): As used herein, the term“umbrella topology blocking agent” refers to an agent which binds to anHA receptor having an umbrella topology glycan. In some embodiments, aUTBA binds to an HA receptor having an umbrella topology glycan found inhuman upper airways. A UBTA can bind to either an umbrella topologyglycan and/or to a cone topology glycan. In some embodiments, a UTBAselectively binds to an umbrella topology glycan with 50, 100, 150, or200% of its affinity for a cone topology glycan. In some embodiments aUTBA selectively binds to an umbrella topology glycan with 50-150% ofits affinity for a cone topology glycan. In some embodiments, and insome embodiments a UTBA binds to an umbrella topology glycan with aboutthe same affinity as for a cone topology glycan. For example, in someembodiments, a UTBA binds an umbrella topology glycan (e.g., 6′SLN-LN)with about 50-200%, 50-150%, or about the same affinity to which itbinds a cone topology glycan (e.g., 3′SLN-LN). In some embodiments, aUTBA selectively inhibits the binding of an influenza virus particle(e.g., a human or avian influenza virus) to the HA receptor based on theglycan topology of the receptor (e.g., umbrella or cone). In someembodiments, a UTBA is a polypeptide. In some such embodiments, a UTBApolypeptide has an amino acid sequence that is substantially identicalor substantially homologous to that of a naturally-occurringpolypeptide. In some embodiments, a UTBA polypeptide is an HApolypeptide. In some embodiments, a UTBA polypeptide is anaturally-occurring HA polypeptide, or a fragment thereof. In someembodiments, a UTBA polypeptide has an amino acid sequence that is notrelated to that of an HA polypeptide. In some embodiments, a UTBApolypeptide is an antibody or fragment thereof. In some embodiments, aUTBA polypeptide is a lectin (e.g., SNA-1). In some embodiments, a UTBAis not a polypeptide. In some embodiments, a UTBA is a small molecule.In some embodiments, a UTBA is a nucleic acid.

Umbrella topology glycan mimic: An “umbrella topology glycan mimic” isan agent, other than an umbrella topology glycan, that binds to bindingagents as described herein. In some embodiments, umbrella topologyglycan mimics are agents that bind to HA polypeptides. In some suchembodiments, umbrella topology glycan mimics are agents that interactwith HA polypeptide residues selected from the group consisting ofresidues 95, 98, 128, 130, 131, 132, 133, 135, 136, 137, 138, 145, 153,155, 156, 158, 159, 160, 183, 186, 187, 188, 189, 190, 192, 193, 194,195, 196, 219, 221, 222, 224, 225, 226, 227, 228 and combinationsthereof. In some such embodiments, umbrella topology glycan mimics areagents that interact with HA polypeptide residues selected from thegroup consisting of residues 130, 131, 132, 133, 135, 137, 155, 188,192, 193, 221, 226, 227, 228, and combinations thereof. In some suchembodiments, umbrella topology glycan mimics are agents that interactwith HA polypeptide residues selected from the group consisting ofresidues 160, 192, 193, and combinations thereof. Note that amino acidpositions stated above are based on H3 HA numbering. In someembodiments, an HA topology glycan mimic is an agent that competes withumbrella topology glycans for interaction with an HA polypeptide.

Umbrella topology specific blocking agent (UTSBA): As used herein, theterm “umbrella topology specific blocking agent” refers to an agentwhich binds to an HA receptor having an umbrella topology glycan foundin human upper airways. A UTSBA selectively binds an umbrella topologyglycan HA. For example, a UTSBA binds an umbrella topology glycan (e.g.,6′SLN-LN) with about at least 2, 4, 5, or 10 times greater affinity thanit binds to a cone topology glycan (e.g., 3′SLN-LN). Typically, theaffinity of a UTSBA for an umbrella topology glycan is greater than 1nM. Typically the affinity of a UTSBA for a cone topology glycan is lessis at least within 2 to 3 orders of magnitude of the binding affinity ofumbrella topology glycans to human adapted HAs such as SC18, Mos99,Tx91, etc. and α2-6 binding plant lectins such as SNA-I. The bindingaffinity of UTSBA as measured by the dose-dependent direct binding assay(FIGS. 19 and 20) would typically be at least 1 nM. Typically theaffinity of a UTSBA for a cone topology glycan is at most 1 to 3 ordersof magnitude less than the binding affinity of cone topology glycans toavian HAs such as Viet0405, Av18, etc. In some embodiments, a UTSBAselectively inhibits binding of an influenza virus particle (e.g., ahuman or avian influenza virus) to the HA receptor (e.g., an H1, H2 orH3 or a human-adapted H5, H7 or H9) based on glycan topology (e.g.,umbrella or cone). In some embodiments, a UTSBA is a polypeptide. Insome such embodiments, a UTSBA polypeptide has an amino acid sequencethat is that is substantially identical or substantially homologous tothat of a naturally-occurring polypeptide. In some embodiments, a UTSBApolypeptide is an HA polypeptide. In some embodiments, a UTSBApolypeptide is a naturally-occurring HA polypeptide, or a fragmentthereof. In some embodiments, a UTSBA polypeptide has an amino acidsequence that is not related to that of an HA polypeptide. In someembodiments, a UTSBA polypeptide is an antibody or fragment thereof. Insome embodiments, a UTSBA polypeptide is a lectin (e.g., SNA-1). In someembodiments, a UTSBA is not a polypeptide. In some embodiments, a UTSBAis a small molecule. In some embodiments, a UTSBA is a nucleic acid.

Vaccination: As used herein, the term “vaccination” refers to theadministration of a composition intended to generate an immune response,for example to a disease-causing agent. For the purposes of the presentinvention, vaccination can be administered before, during, and/or afterexposure to a disease-causing agent, and/or to the development of one ormore symptoms, and in some embodiments, before, during, and/or shortlyafter exposure to the agent. In some embodiments, vaccination includesmultiple administrations, appropriately spaced in time, of a vaccinatingcomposition.

Variant: As used herein, the term “variant” is a relative term thatdescribes the relationship between a particular polypeptide (e.g., HApolypeptide) of interest and a “parent” polypeptide to which itssequence is being compared. A polypeptide of interest is considered tobe a “variant” of a parent polypeptide if the polypeptide of interesthas an amino acid sequence that is identical to that of the parent butfor a small number of sequence alterations at particular positions.Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% ofthe residues in the variant are substituted as compared with the parent.In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substituted residue as compared with a parent. Often, a variant has avery small number (e.g., fewer than 5, 4, 3, 2, or 1) number ofsubstituted functional residues (i.e., residues that participate in aparticular biological activity). Furthermore, a variant typically hasnot more than 5, 4, 3, 2, or 1 additions or deletions, and often has noadditions or deletions, as compared with the parent. Moreover, anyadditions or deletions are typically fewer than about 25, about 20,about 19, about 18, about 17, about 16, about 15, about 14, about 13,about 10, about 9, about 8, about 7, about 6, and commonly are fewerthan about 5, about 4, about 3, or about 2 residues. In someembodiments, the parent polypeptide is one found in nature. For example,a parent HA polypeptide may be one found in a natural (e.g., wild type)isolate of an influenza virus (e.g., a wild type HA).

Vector: As used herein, “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. In some embodiment, vectors are capable of extra-chromosomalreplication and/or expression of nucleic acids to which they are linkedin a host cell such as a eukaryotic or prokaryotic cell. Vectors capableof directing the expression of operatively linked genes are referred toherein as “expression vectors.”

Wild type: As is understood in the art, the phrase “wild type” generallyrefers to a normal form of a protein or nucleic acid, as is found innature. For example, wild type HA polypeptides are found in naturalisolates of influenza virus. A variety of different wild type HAsequences can be found in the NCBI influenza virus sequence database,http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html. Certain exemplary wildtype H2 HA polypeptides are presented in FIG. 1.

DETAILED DESCRIPTION OF CERTAIN PARTICULAR EMBODIMENTS OF THE INVENTION

The present invention provides binding agents that show a strong abilityto discriminate between umbrella-topology and cone-topology glycans. Insome embodiments, provided binding agents are engineered HApolypeptides. In some embodiments, provided binding agents areengineered H2 HA polypeptides. In some embodiments, provided bindingagents show an ability to discriminate between umbrella-topology andcone-topology glycans that is at least effective as that shown by anRTLS HA polypeptide (e.g., an RTLS H2 HA polypeptide) as describedherein.

The present invention also provides systems and reagents for identifyingbinding agents that show a strong ability to discriminate betweenumbrella-topology and cone-topology glycans. The present invention alsoprovides various reagents and methods associated with provided bindingagents including, for example, systems for identifying them, strategiesfor preparing them, antibodies that bind to them, and various diagnosticand therapeutic methods relating to them. Further description of certainembodiments of these aspects, and others, of the present invention, ispresented below.

Hemagglutinin (HA)

Influenza viruses are RNA viruses which are characterized by a lipidmembrane envelope containing two glycoproteins, hemagglutinin (HA) andneuraminidase (NA), embedded in the membrane of the virus particular.There are 16 known HA subtypes and 9 NA subtypes, and differentinfluenza strains are named based on the number of the strain's HA andNA subtypes. Based on comparisons of amino acid sequence identity and ofcrystal structures, the HA subtypes have been divided into two maingroups and four smaller clades. The different HA subtypes do notnecessarily share strong amino acid sequence identity, but the overall3D structures of the different HA subtypes are similar to one another,with several subtle differences that can be used for classificationpurposes. For example, the particular orientation of the membrane-distalsubdomains in relation to a central α-helix is one structuralcharacteristic commonly used to determine HA subtype (Russell et al.,2004 Virology, 325:287, 2004; incorporated herein by reference).

HA exists in the membrane as a homotrimer of one of 16 subtypes, termedH1-H16. Only three of these subtypes (H1, H2, and H3) have thus farbecome adapted for human infection. One reported characteristic of HAsthat have adapted to infect humans (e.g., of HAs from the pandemic H1N1(1918) and H3N2 (1967-68) influenza subtypes) is their ability topreferentially bind to α2-6 sialylated glycans in comparison with theiravian progenitors that preferentially bind to α2-3 sialylated glycans(Skehel & Wiley, 2000 Annu Rev Biochem, 69:531; Rogers, & Paulson, 1983Virology, 127:361; Rogers et al., 1983 Nature, 304:76; Sauter et al.,1992 Biochemistry, 31:9609; Connor et al., 1994 Virology, 205:17; Tumpeyet al., 2005 Science, 310:77; all of which are incorporated herein byreference). The present invention, however, encompasses the recognitionthat ability to infect human hosts correlates less with binding toglycans of a particular linkage, and more with binding to glycans of aparticular topology. Thus, the present invention demonstrates that HAsthat mediate infection of humans bind to umbrella topology glycans,often showing preference for umbrella topology glycans over conetopology glycans (even though cone-topology glycans may be α2-6sialylated glycans).

Several crystal structures of HAs from H1 (human and swine), H3 (avian)and H5 (avian) subtypes bound to sialylated oligosaccharides (of bothα2-3 and α2-6 linkages) are available and provide molecular insightsinto the specific amino acids that are involved in distinct interactionsof the HAs with these glycans (Eisen et al., 1997 Virology, 232:19; Haet al., 2001 Proc Natl Acad Sci USA, 98:11181; Ha et al., 2003 Virology,309:209; Gamblin et al., 2004 Science, 303:1838; Stevens et al., 2004Science, 303:1866; Russell et al., 2006 Glycoconj J 23:85; Stevens etal., 2006 Science, 312:404; all of which are incorporated herein byreference).

For example, the crystal structures of H5 (A/duck/Singapore/3/97) aloneor bound to an α2-3 or an α2-6 sialylated oligosaccharide identifiescertain amino acids that interact directly with bound glycans, and alsoamino acids that are one or more degree of separation removed (Stevenset al., 2001 Proc Natl Acad Sci USA 98:11181; incorporated herein byreference). In some cases, conformation of these residues is differentin bound versus unbound states. For instance, Glu190, Lys193 and Gln226all participate in direct-binding interactions and have differentconformations in the bound versus the unbound state. The conformation ofAsn186, which is proximal to Glu190, is also significantly different inthe bound versus the unbound state.

Crystal structures of exemplary H2 HAs (human viruses A/Singapore/1/57and A/Japan/305/57, avian viruses A/ck/Postdam/84, A/dk/Ontario/77 andA/ck/NewYork/91) complexed with analogs of human and avian HA receptorsidentify certain amino acids that interact directly with bound glycansand also mutations that alter the receptor binding pocket of HA (Xu R etal., 2010 J Virol 84(4):1715; Liu J, et al., 2009 Proc Natl Acad Sci USA106(40):17175; each of which is incorporated herein by reference).Certain secondary structure elements of the binding site, e.g., the 130-and 220-loops and/or the 190-helix, may affect interactions with humanand/or avian receptors. For example, human H2 HA residue 222 (Lys) formsa hydrogen bond with the 3′OH of Gal-2; human H2 HA residue 226(leucine) is reported to lead to a more hydrophobic environment thanthat prevent in avian HA's (Liu J, et al., 2009 Proc Natl Acad Sci USA106(40):17175). It has been reported that the receptor-binding site isformed by a shallow cavity surrounded by residues from the 190 helix(residues 190 to 198), the 220 loop (residues 221 to 228), the 130 loop(residues 134 to 138), and Thr¹⁵⁵ (Xu R et al., 2010 J Virol84(4):1715). It has been observed that several conserved aromaticresidues, including Tyr⁹⁸, Trp¹⁵³, and His¹⁸³, may form the bottom ofthe depression of the receptor-binding site (Xu R et al., 2010 J Virol84(4):1715). In some embodiments, a sequence motif V/I H H P is presentin the H2 HA receptor binding site, where the first H corresponds to ahistidine at Residue 183. In some such embodiments, a glycine may bepresent at Residue 134, a tryptophan may be present at Residue 153, athreonine may be present at residue 155, a glutamic acid may be presentat Residue 190, and/or a leucine may be present at Residue 194, andcombinations thereof. In some embodiments, Residues 134, 153, 155, 190and 194 are involved in binding to sialic acid.

Binding Agents

As described herein, binding to umbrella topology glycans correlateswith ability to mediate infection of particular hosts, including forexample, humans. Accordingly, the present invention provides bindingagents (e.g., HA polypeptides, particularly H2 HA polypeptides, LSBAs,UTBAs, UTSBAs, etc.) that bind to umbrella glycans (and/or to umbrellatopology glycan mimics). In some embodiments, inventive binding agentsbind to umbrella glycans (and/or to umbrella topology glycan mimics)with high affinity. In some embodiments, inventive binding agents bindto a plurality of different umbrella topology glycans, often with highaffinity and/or specificity.

In some embodiments, inventive binding agents bind to umbrella topologyglycans (e.g., long α2-6 silaylated glycans such as, for example,Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc-) with high affinity. Forexample, in some embodiments, inventive binding agents bind to umbrellatopology glycans with an affinity comparable to that observed for a wildtype HA that mediates infection of a humans. In some embodiments, a wildtype HA that mediates infection in humans (e.g., is human transmissible)is an H1N1 HA, H2N2 HA, and/or H3N2 HA. In some embodiments, a wild typeHA that mediates infection in humans (e.g., is human transmissible) isan HA from A/South Carolina/1/1918. In some embodiments, a wild type HAthat mediates infection in humans (e.g., is human transmissible) is anHA from A/Albany/6/58. In some embodiments, inventive binding agentsbind to umbrella glycans within a range of 10-fold or less (e.g.,9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold,1.5-fold, etc.) of the affinity for a wild type HA that mediatesinfection of a humans

In some embodiments, inventive binding agents bind to umbrella glycanswith an affinity that is at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 100% of that observed under comparable conditionsfor a wild type HA that mediates infection of humans (e.g., is humantransmissible). In some embodiments, inventive binding agents bind toumbrella glycans with an affinity that is greater than that observedunder comparable conditions for a wild type HA that mediates infectionof humans (e.g., is human transmissible).

In some embodiments, binding affinity of inventive binding agents isassessed over a range of concentrations. Such a strategy providessignificantly more information, particularly in multivalent bindingassays, than do single-concentration analyses. In some embodiments, forexample, binding affinities of inventive binding agents are assessedover concentrations ranging over at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10 ormore fold.

In some embodiments, binding partner concentration (e.g., HA receptor,glycan, etc.) may be fixed to be in excess of ligand (e.g., an HApolypeptide) concentration so as to mimic physiological conditions(e.g., viral HA binding to cell surface glycans). Alternatively oradditionally, in some embodiments, binding partner (e.g., HA receptor,glycan, etc.) concentration and/or ligand (e.g., an HA polypeptide)concentration may be varied. In some such embodiments, affinity (e.g.,binding affinity) may be compared to a reference (e.g., a wild type HAthat mediates infection of a humans) under comparable conditions (e.g.,concentrations).

In some embodiments, binding affinity of inventive binding agents isperformed using whole viruses. In some such embodiments, viral titer ismeasured in units that directly correlate with the number of viralparticles.

In some embodiments, inventive binding agents show high affinity if theyshow a saturating signal in a multivalent glycan array binding assaysuch as those described herein. In some embodiments, inventive bindingagents show high affinity if they show a signal above about 400000 ormore (e.g., above about 500000, about 600000, about 700000, about800000, etc) in such studies. In some embodiments, binding agents asdescribed herein show saturating binding to umbrella glycans over aconcentration range of at least 2 fold, at least 3 fold, at least 4fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70fold, at least 80 fold, at least 90 fold, at least 100 fold or more, andin some embodiments over a concentration range as large as at least 200fold or more.

In some embodiments, provided binding agents show high affinity bindingto umbrella topology glycans (and/or to umbrella topology glycanmimics). In some embodiments, provided binding agents show an affinity(Kd′) for umbrella-topology glycans within the range of about 1.5 nM toabout 2 pM. In some embodiments, provided binding agents show anaffinity (Kd′) for umbrella-topology glycans within the range of about1.5 nM to about 200 pM. In some embodiments, provided binding agentsshow an affinity (Kd′) for umbrella-topology glycans within the range ofabout 200 pM to about 10 pM. In some embodiments, provided bindingagents show an affinity (Kd′) for umbrella-topology glycans within therange of about 10 pM to about 2 pM. In some embodiments, providedbinding agents show high affinity binding to umbrella topology glycans(and/or to umbrella topology glycan mimics) if they show a Kd′ of about500 pM or less (e.g., below about 400 pM, about 300 pM, about 200 pM,about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about50 pM, about 40 pM, about 30 pM, about 20 pM, about 10 pM, about 5 pM,about 4 pM, about 3 pM, about 2 pM, etc.) in binding assays.

In some embodiments, provided binding agents show low affinity bindingto cone topology glycans (and/or to cone topology glycan mimics). Insome embodiments, provided binding agents show low affinity binding tocone topology glycans (and/or to cone topology glycan mimics) if theyshow a Kd′ of about 100 pM or more (e.g., above about 200 pM, about 300pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800pM, about 900 pM, about 1 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM,about 1.4 nM, about 1.5 nM, etc.) in binding assays.

In some embodiments, provided binding agents show both high affinity toumbrella topology glycans (and/or to umbrella topology glycan mimics)and low affinity to cone topology glycans (and/or to cone topologyglycan mimics). In some embodiments, provided binding agents show a Kd′of about 500 pM or less (e.g., below about 400 pM, about 300 pM, about200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60pM, about 50 pM, about 40 pM, about 30 pM, about 20 pM, about 10 pM,about 5 pM, about 4 pM, about 3 pM, about 2 pM, etc.) for umbrellatopology glycans and a Kd′ of about 100 pM or more (e.g., above about200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about700 pM, about 800 pM, about 900 pM, about 1 nM, about 1.1 nM, about 1.2nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, etc.) for cone topologyglycans in binding assays.

In one aspect, the present invention provides the surprising recognitionthat high affinity for umbrella topology glycans, alone, may not besufficient to mediate effective and/or efficient transmission to humans.Rather, according to the present disclosure, in some embodiments,provided binding agents show low binding to cone topology glycans and/orboth high affinity for umbrella-topology glycans and low affinity forcone-topology glycans.

In some embodiments, inventive binding agents bind to α2-6 sialylatedglycans; in some embodiments, inventive binding agents bindpreferentially to α2-6 sialylated glycans. In some embodiments,inventive binding agents bind to a plurality of different α2-6sialylated glycans. In some embodiments, inventive binding agents arenot able to bind to α2-3 sialylated glycans, and in other embodimentsinventive binding agents are able to bind to α2-3 sialylated glycans.

Furthermore, in some embodiments, inventive binding agentspreferentially bind to umbrella topology glycans (and/or to umbrellatopology glycan mimics) (e.g., they bind more strongly) than they bindto cone topology glycans. In some embodiments, inventive binding agentsshow a relative affinity for umbrella glycans vs cone glycans that isabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 20, about 30, about 40, about 50, about 60,about 70, about 80, about 90, about 100, about 200, about 300, about400, about 500, about 600, about 700, about 800, about 900, about 1000,about 2000, about 3000, about 4000, about 5000, about 6000, about 7000,about 8000, about 9000, about 10,000, up to about 100,000 or more. Insome embodiments, inventive binding agents show an affinity for umbrellatopology glycans that is about 100%, about 200%, about 300%, about 400%,about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%,about 2000%, about 3000%, about 4000%, about 5000%, about 6000%, about7000%, about 8000%, about 9000%, about 10,000% or more than theiraffinity for cone topology glycans.

In some embodiments, inventive binding agents bind to receptors found onhuman upper respiratory epithelial cells. In some embodiments, inventivebinding agents bind to HA receptors in the bronchus and/or trachea. Insome embodiments, inventive binding agents are not able to bindreceptors in the deep lung, and in other embodiments, inventive bindingagents are able to bind receptors in the deep lung.

In some embodiments, inventive binding agents bind to at least about10%, about 15%, about 20%, about 25%, about 30% about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95% or more of the glycansfound on HA receptors in human upper respiratory tract tissues (e.g.,epithelial cells).

In some embodiments, inventive binding agents bind to one or more of theglycans illustrated in FIG. 4. In some embodiments, inventive bindingagents bind to multiple glycans illustrated in FIG. 4. In someembodiments, inventive binding agents bind with high affinity and/orspecificity to glycans illustrated in FIG. 4. In some embodiments,inventive binding agents bind to glycans illustrated in FIG. 4preferentially as compared with their binding to glycans illustrated inFIG. 3. In some embodiments, inventive binding agents bind to anoligosaccharide of the following form:

Neu5Acα2-6Sug1-Sug2-Sug3

where:

-   -   1. Neu5Ac α2-6 is always or almost always at the non-reducing        end;    -   2. Sug1:        -   a. is a hexose (frequently Gal or Glc) or hexosamine (GlcNAc            or GalNAc) in α or β configuration (frequently β- for N- and            O-linked extension and α- in the case of GalNAcα- that is            O-linked to glycoprotein);        -   b. no sugars other than Neu5Acα2-6 should be attached to any            of the non-reducing positions of Sug1 (except when Sug1 is            GalNAcα- that is O-linked to the glycoprotein); and/or        -   c. non-sugar moieties such as sulfate, phosphate, guanidium,            amine, N-acetyl, etc. can be attached to non-reducing            positions (typically 6 position) of Sug1 to improve contacts            with HA;    -   3. Sug2 and/or Sug3:        -   a. hexose (frequently Gal or Glc) or hexosamine (GlcNAc or            GalNAc) in α or β configuration (frequently β); and/or        -   b. sugars (such as Fuc) or non-sugar moieties such as            sulfate, phosphate, guanidium, amine, N-acetyl, etc. can be            attached to non-reducing positions of Sug2, Sug3, and/or            Sug4;    -   4. Linkage between any two sugars in the oligosaccharide apart        from Neu5Acα2-6 linkage can be 1-2, 1-3, 1-4, and/or 1-6        (typically 1-3 or 1-4); and/or    -   5. Structure where Neu5Acα2-6 is linked GalNAcα that is O-linked        to the glycoprotein and additional sugars are linked to the        non-reducing end of GalNAcα for example

i. Neu5Acα2-6(Neu5Acα2-3Galβ1-3)GalNAcα- ii. Neu5Acα2-6(Galβ1-3)GalNAcα-

The present invention provides binding agents with designated bindingspecificity, and also provides binding agents with designated bindingcharacteristics with respect to umbrella glycans.

Certain particular binding agents provided by the present invention aredescribed in more detail below.

HA Polypeptides

In some embodiments, inventive binding agents are HA polypeptides. Forexample, the present invention provides isolated HA polypeptides withdesignated binding specificity, and also provides engineered HApolypeptides with designated binding characteristics with respect toumbrella glycans.

In some embodiments, provided HA polypeptides with designated bindingcharacteristics are H1 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H2 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H3 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H4 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H5 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H6 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H7 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H8 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H9 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H10 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H11 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H12 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H13 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H14 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H15 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H16 polypeptides.

In some embodiments, HA polypeptides in accordance with the inventionwith designated binding characteristics are not H1 polypeptides, are notH2 polypeptides, and/or are not H3 polypeptides.

In some embodiments, HA polypeptides in accordance with the invention donot include the H1 protein from any of the strains: A/SouthCarolina/1/1918; A/Puerto Rico/8/1934; A/Taiwan/1/1986; A/Texas/36/1991;A/Beijing/262/1995; A/Johannesburg/92/1996; A/New Calcdonia/20/1999;A/Solomon Islands/3/2006.

In some embodiments, HA polypeptides in accordance with the inventionare not the H2 protein from any of the strains of the Asian flu epidemicof 1957-58). In some embodiments, HA polypeptides in accordance with theinvention do not include the H2 protein from any of the strains:A/Japan/305+/1957; A/Singapore/1/1957; A/Taiwan/1/1964; A/Taiwan/1/1967.In some embodiments, HA polypeptides in accordance with the invention donot include the H2 protein from A/Chicken/Pennsylvania/2004.

In some embodiments, HA polypeptides in accordance with the invention donot include the H3 protein from any of the strains: A/Aichi/2/1968;A/Philipines/2/1982; A/Mississippi/1/1985; A/Leningrad/360/1986;A/Sichuan/2/1987; A/Shanghai/11/1987; A/Beijing/353/1989;A/Shandong/9/1993; A/Johannesburg/33/1994; A/Nanchang/813/1995;A/Sydney/5/1997; A/Moscow/10/1999; A/Panama/2007/1999; A/Wyoming/3/2003;A/Oklahoma/323/2003; A/California/7/2004; A/Wisconsin/65/2005.

Engineered and/or Variant HA Polypeptides

In some embodiments, a provided HA polypeptide is a variant of a parentHA polypeptide in that its amino acid sequence is identical to that ofthe parent HA but for a small number of particular sequence alterations.In some embodiments, the parent HA is an HA polypeptide found in anatural isolate of an influenza virus (e.g., a wild type HApolypeptide). In some embodiments, the parent HA is an H2 HApolypeptide. In some embodiments, the parent HA is a wild-type H2 HApolypeptide. In some embodiments, the parent HA is an H2 HA selectedfrom the group listed in FIG. 1. In some such embodiments, the parent HAis CkPA04. In some embodiments, the parent HA is Alb58. In someembodiments, the parent HA is A/Singapore/1/57 or A/Japan/305/57. Insome embodiments, the parent HA is A/ck/NewYork/29878/91,A/dk/Ontario/77 or A/ck/postdam/4705/84.

In some embodiments, inventive HA polypeptide variants have differentglycan binding characteristics than their corresponding parent HApolypeptides. In some embodiments, inventive HA variant polypeptideshave greater affinity and/or specificity for umbrella glycans (e.g., ascompared with for cone glycans) than do their cognate parent HApolypeptides. In some embodiments, such HA polypeptide variants areengineered variants.

In some embodiments, HA polypeptide variants with altered glycan bindingcharacteristics have sequence alternations in residues within oraffecting the glycan binding site. In some embodiments, suchsubstitutions are of amino acids that interact directly with boundglycan; in other embodiments, such substitutions are of amino acids thatare one degree of separation removed from those that interact with boundglycan, in that the one degree of separation removed-amino acids either(1) interact with the direct-binding amino acids; (2) otherwise affectthe ability of the direct-binding amino acids to interact with glycan,but do not interact directly with glycan themselves; or (3) otherwiseaffect the ability of the direct-binding amino acids to interact withglycan, and also interact directly with glycan themselves. Inventive HApolypeptide variants contain substitutions of one or more direct-bindingamino acids, one or more first degree of separation-amino acids, one ormore second degree of separation-amino acids, or any combination ofthese. In some embodiments, inventive HA polypeptide variants maycontain substitutions of one or more amino acids with even higherdegrees of separation.

In some embodiments, HA polypeptide variants with altered glycan bindingcharacteristics have sequence alterations in residues that make contactwith sugars beyond Neu5Ac and Gal (see, for example, FIG. 13).

In some embodiments, HA polypeptide variants have at least one aminoacid substitution, as compared with a wild type parent HA. In someembodiments, inventive HA polypeptide variants have at least two, three,four, five or more amino acid substitutions as compared with a cognatewild type parent HA; in some embodiments inventive HA polypeptidevariants have two, three, or four amino acid substitutions. In someembodiments, all such amino acid substitutions are located within theglycan binding site.

In some embodiments, an HA polypeptide variant, and particularly an H2polypeptide variant has one or more amino acid substitutions relative toa wild type parent HA at residues selected from amino acids that are onedegree of separation removed from those that interact with bound glycan,in that the one degree of separation removed-amino acids either (1)interact with the direct-binding amino acids; (2) otherwise affect theability of the direct-binding amino acids to interact with glycan, butdo not interact directly with glycan themselves; or (3) otherwise affectthe ability of the direct-binding amino acids to interact with glycan,and also interact directly with glycan themselves, including but notlimited to residues 137, 145, 156, 159, 186, 187, 189, 190, 192, 193,196, 222, 225, 226, and 228.

In some embodiments, HA polypeptide variants, and particularly H2polypeptide variants, have sequence substitutions at positionscorresponding to one or more of residues 137, 193, 226, and 228.Alternatively or additionally, in some embodiments, HA polypeptidevariants have sequence substitutions at positions corresponding to oneor more of residues 145, 156, 159, 186, 187, 189, 190, 192, 196, 222 and225.

In some embodiments, provided HA polypeptides such as HA polypeptidevariants (e.g., H2 HA polypeptides such as H2 HA polypeptide variants)have an amino acid residue at a position corresponding to 137 (a“Residue 137”) that is selected from arginine, lysine, glutamine,methionine and histidine. In some embodiments, provided HA polypeptidessuch as HA polypeptide variants (e.g., H2 HA polypeptides such as H2 HApolypeptide variants) have an amino acid residue at a positioncorresponding to 137 (a “Residue 137”) that is selected from arginine,lysine, glutamine, and methionine. In some embodiments, provided HApolypeptides such as HA polypeptide variants (e.g., H2 HA polypeptidessuch as H2 HA polypeptide variants) have an amino acid residue at aposition corresponding to 137 (a “Residue 137”) that is selected fromarginine and lysine. In some embodiments, provided HA polypeptidesand/or polypeptide variants (e.g., H2 HA polypeptide variants) have anarginine residue as Residue 137.

In some embodiments, provided HA polypeptides such as HA polypeptidevariants (e.g., H2 HA polypeptides such as H2 HA polypeptide variants)have a an amino acid residue at a position corresponding to 193(“Residue 193”) that is selected from the group consisting of alanine,aspartic acid, glutamic acid, leucine, isoleucine, methionine, serine,threonine, cysteine, and valine. In some embodiments, provided HApolypeptides such as HA polypeptide variants (e.g., H2 HA polypeptidessuch as H2 HA polypeptide variants) have a Residue 193 that is selectedfrom the group consisting of alanine, glutamic acid, threonine,cysteine, methionine, valine, and serine. In some embodiments, providedHA polypeptides such as HA polypeptide variants (e.g., H2 HApolypeptides such as H2 HA polypeptide variants) have a Residue 193 thatis selected from the group consisting of alanine, glutamic acid andthreonine. In some embodiments, provided HA polypeptides such as HApolypeptide variants (e.g., H2 HA polypeptides such as H2 HA polypeptidevariants) have a Residue 193 that is threonine.

In some embodiments, provided HA polypeptides such as HA polypeptidevariants (e.g., H2 HA polypeptides such as H2 HA polypeptide variants)have an amino acid as a residue corresponding to residue 226 (“Residue226”) that is a nonpolar amino acid. In some embodiments, provided HApolypeptides such as HA polypeptide variants (e.g., H2 HA polypeptidessuch as H2 HA polypeptide variants) have a Residue 226 that is selectedfrom the group consisting of alanine, cysteine, glycine, isoleucine,leucine, methionine, phenylalanine, proline, tryptophan and valine. Insome embodiments, provided HA polypeptides such as HA polypeptidevariants (e.g., H2 HA polypeptides such as H2 HA polypeptide variants)have a Residue 226 that is selected from the group consisting ofleucine, isoleucine and valine. In some embodiments, provided HApolypeptides such as HA polypeptide variants (e.g., H2 HA polypeptidessuch as H2 HA polypeptide variants) have a Residue 226 that is leucine.

In some embodiments, provided HA polypeptides such as HA polypeptidevariants (e.g., H2 HA polypeptides such as H2 HA polypeptide variants)have an amino acid residue at a position corresponding to 228 (“Residue228”) that is a polar amino acid. In some embodiments, provided HApolypeptides such as HA polypeptide variants (e.g., H2 HA polypeptidessuch as H2 HA polypeptide variants) have a Residue 228 that is selectedfrom the group consisting of arginine, asparagine, aspartic acid,glutamic acid, glutamine, histidine, lysine, serine, glycine, threonine,and tyrosine. In some embodiments, provided HA polypeptides such as HApolypeptide variants (e.g., H2 HA polypeptides such as H2 HA polypeptidevariants) have Residue 228 that is selected from the group consisting ofarginine, asparagine, serine, glycine, and threonine. In someembodiments, provided HA polypeptides such as HA polypeptide variants(e.g., H2 HA polypeptides such as H2 HA polypeptide variants) have aResidue 228 that is serine.

In some embodiments, provided HA polypeptide variants have at least onesubstitution in a position other than 137, 193, 226, and/or 228, ascompared with a particular reference HA polypeptide (e.g., with a wildtype HA polypeptide such as a wild type H2 HA polypeptide, for exampleas described herein). In some such embodiments, affinity and/orspecificity of the variant for umbrella-topology glycans is increased.

In some embodiments, provided HA polypeptides such as HA polypeptidevariants (e.g., H2 HA polypeptides such as H2 HA polypeptide variants)have an amino acid at a particular residue (e.g., 137, 145, 186, 187,189, 190, 192, 193, 222, 225, 226, 228) that is predominantly present inthe corresponding human-adapted HA (e.g., human-adapted H2 HA, such asthose shown in FIG. 1). In some embodiments, provided HA polypeptidessuch as HA polypeptide variants (e.g., H2 HA polypeptides such as H2 HApolypeptide variants) have at least one amino acid substitution that isfound in the corresponding human-adapted HA (e.g., human-adapted H2 HA).

In some embodiments, inventive HA polypeptide variants have an openbinding site as compared with a reference or parent HA, and particularlywith a parent wild type HAs.

Portions or Fragments of HA Polypeptides

The present invention further provides characteristic portions (whichmay or may not be binding agents) of HA polypeptides in accordance withthe invention (and/or polypeptide variants) and nucleic acids thatencode them. In general, a characteristic portion is one that contains acontinuous stretch of amino acids, or a collection of continuousstretches of amino acids, that together are characteristic of the HApolypeptide. Each such continuous stretch generally will contain atleast two amino acids. Furthermore, those of ordinary skill in the artwill appreciate that typically at least 5, 10, 15, 20 or more aminoacids are required to be characteristic of an HA polypeptide (e.g., anH2 HA polypeptide). In general, a characteristic portion is one that, inaddition to the sequence identity specified above, shares at least onefunctional characteristic with the relevant intact HA polypeptide. Insome embodiments, inventive characteristic portions of HA polypeptidesshare glycan binding characteristics with the relevant full-length HApolypeptides.

Non-HA Polypeptides

In some embodiments, binding agents provided in accordance with thepresent invention are polypeptides whose amino acid sequence does notinclude a characteristic HA sequence. Such polypeptides are referred toherein as “Non-HA polypeptides”. In some embodiments, a Non-HApolypeptide has an amino acid sequence selected in advance (e.g., viarational design, including for example, introduction of strategic aminoacid alterations [additions, deletions, and/or substitutions] ascompared with a reference sequence). In some embodiments, a Non-HApolypeptide has an amino acid sequence that is determined stochasticallyand, for example, identified on the basis of the desirable bindingcharacteristics defined herein.

Antibodies

In some embodiments, binding agents provided in accordance with thepresent invention are antibodies (e.g., that bind to umbrella topologyglycans and/or to umbrella topology glycan mimics). Antibodies suitablefor the invention include antibodies or fragments of antibodies thatbind immunospecifically to any umbrella topology glycan epitope. As usedherein, the term “antibodies” is intended to include immunoglobulins andfragments thereof which are specifically reactive to the designatedprotein or peptide, or fragments thereof. Suitable antibodies include,but are not limited to, human antibodies, primatized antibodies,chimeric antibodies, bi-specific antibodies, humanized antibodies,conjugated antibodies (i.e., antibodies conjugated or fused to otherproteins, radiolabels, cytotoxins), Small Modular ImmunoPharmaceuticals(“SMIPs™”), single chain antibodies, cameloid antibodies, and antibodyfragments. As used herein, the term “antibodies” also includes intactmonoclonal antibodies, polyclonal antibodies, single domain antibodies(e.g., shark single domain antibodies (e.g., IgNAR or fragmentsthereof)), multispecific antibodies (e.g. bi-specific antibodies) formedfrom at least two intact antibodies, and antibody fragments so long asthey exhibit the desired biological activity. Antibody polypeptides foruse herein may be of any type (e.g., IgA, IgD, IgE, IgG, IgM).

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; triabodies; tetrabodies; linear antibodies;single-chain antibody molecules; and multi specific antibodies formedfrom antibody fragments. The term “antibody fragment” also includes anysynthetic or genetically engineered protein that acts like an antibodyby binding to a specific antigen to form a complex. For example,antibody fragments include isolated fragments, “Fv” fragments,consisting of the variable regions of the heavy and light chains,recombinant single chain polypeptide molecules in which light and heavychain variable regions are connected by a peptide linker (“ScFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

Antibodies can be generated using methods well known in the art. Forexample, protocols for antibody production are described by Harlow andLane, 1988, Antibodies: A Laboratory Manual; incorporated herein byreference. Typically, antibodies can be generated in mouse, rat, guineapig, hamster, camel, llama, shark, or other appropriate host.Alternatively, antibodies may be made in chickens, producing IgYmolecules (Schade et al., 1996 ALTEX 13(5):80; incorporated herein byreference). In some embodiments, antibodies suitable for the presentinvention are subhuman primate antibodies. For example, generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., international patentpublication No. WO 91/11465 (1991), and in Losman et al., 1990 Int. J.Cancer 46: 310; each of which is incorporated herein by reference. Insome embodiments, monoclonal antibodies may be prepared using hybridomamethods (Milstein and Cuello, 1983 Nature 305(5934):537; incorporatedherein by reference). In some embodiments, monoclonal antibodies mayalso be made by recombinant methods (U.S. Pat. No. 4,166,452, 1979;incorporated herein by reference).

In some embodiments, antibodies suitable for the invention may includehumanized or human antibodies. Humanized forms of non-human antibodiesare chimeric Igs, Ig chains or fragments (such as Fv, Fab, Fab′, F(ab′)2or other antigen-binding subsequences of Abs) that contain minimalsequence derived from non-human Ig. Generally, a humanized antibody hasone or more amino acid residues introduced from a non-human source.These non-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization is accomplished by substituting rodent complementaritydetermining regions (CDRs) or CDR sequences for the correspondingsequences of a human antibody (Riechmann et al., 1988 Nature332(6162):323; Verhoeyen et al., 1988 Science. 239(4847):1534; each ofwhich is incorporated herein by reference). Such “humanized” antibodiesare chimeric Abs (U.S. Pat. No. 4,816,567, 1989; incorporated herein byreference), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In some embodiments, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentAbs. Humanized antibodies include human Igs (recipient antibody) inwhich residues from a CDR of the recipient are replaced by residues froma CDR of a non-human species (donor antibody) such as mouse, rat orrabbit, having the desired specificity, affinity and capacity. In someinstances, corresponding non-human residues replace Fv frameworkresidues of the human Ig. Humanized antibodies may comprise residuesthat are found neither in the recipient antibody nor in the imported CDRor framework sequences. In general, the humanized antibody comprisessubstantially all of at least one, and typically two, variable domains,in which most if not all of the CDR regions correspond to those of anon-human Ig and most if not all of the FR regions are those of a humanIg consensus sequence. The humanized antibody optimally also comprisesat least a portion of an Ig constant region (Fc), typically that of ahuman Ig (Riechmann et al., 1988 Nature 332(6162):323; Verhoeyen et al.,1988 Science. 239(4847):1534; each of which is incorporated herein byreference).

Human antibodies can also be produced using various techniques,including phage display libraries (Hoogenboom et al., 1991 Mol. Immunol.28(9):1027-37; Marks et al., 1991 J Mol Biol. 222(3):581-97; each ofwhich is incorporated herein by reference) and the preparation of humanmonoclonal antibodies (Reisfeld and Sell, 1985, Cancer Surv.4(1):271-90; incorporated herein by reference). Similarly, introducinghuman Ig genes into transgenic animals in which the endogenous Ig geneshave been partially or completely inactivated can be exploited tosynthesize human antibodies. Upon challenge, human antibody productionis observed, which closely resembles that seen in humans in allrespects, including gene rearrangement, assembly, and antibodyrepertoire (Fishwild et al., High-avidity human IgG kappa monoclonalantibodies from a novel strain of minilocus transgenic mice, Nat.Biotechnol. 1996 July; 14(7):845-51; Lonberg et al., Antigen-specifichuman antibodies from mice comprising four distinct geneticmodifications, Nature 1994 Apr. 28; 368(6474): 856-9; Lonberg andHuszar, Human antibodies from transgenic mice, Int. Rev. Immunol. 1995;13(1): 65-93; Marks et al., By-passing immunization: building highaffinity human antibodies by chain shuffling. Biotechnology (N Y). 1992July; 10(7):779-83; each of which is incorporated herein by reference).

Lectins

In some embodiments, binding agents provided in accordance with thepresent invention are lectins. Lectins are sugar-binding proteins whichmay bind to a soluble carbohydrate or to a carbohydrate moiety which isa part of a glycoconjugate (e.g., a glycopeptide or glycolipid). Lectinstypically agglutinate certain animal cells and/or precipitateglycoconjugates by recognizing a particular sugar moiety. For example,SNA-1 is a lectin that has a high affinity for α2-6 sialic acids. As yetanother example, polyporus squamosus lectins (PSL1a and PSL1b) have highaffinity for binding sialylated glycoconjugates containingNeu5Acα-2,6Galβ1,4Glc/GlcNAc trisaccharide sequences ofasparagine-linked glycoproteins. Non-limiting exemplary lectins that mayact as binding agents include SNA-1, SNA-1′, PSL1a, PSL1b, andpolypeptides derived therefrom.

Amino acid sequences of exemplary lectins are provided below in Tables1-4.

TABLE 1 Sambucus Nigra Lectin 1 (Genbank Accession No. U27122):(SEQ ID NO: 24) MRLVAKLLYLAVLAICGLGIHGALTHPRVTPPVYPSVSFNLTGADTYEPFLRALQEKVILGNHTAFDLPVLNPESQVSDSNRFVLVPLTNPSGDTVTLAIDVVNLYVVAFSSNGKSYFFSGSTAVQRDNLFVDTTQEELNFTGNYTSLERQVGFGRVYIPLGPKSLDQAISSLRTYTLTAGDTKPLARGLLVVIQMVSEAARFRYIELRIRTSITDASEFTPDLLMLSMENNWSSMSSEIQQAQPGGIFAGVVQLRDERNNSIEVTNFRRLFELTYIAVLLYGCAPVTSSSYSNNAIDAQIIKMPVFRGGEYEKVCSVVEVTRRISGWDGLCVDVRYGHYIDGNPVQLRPCGNECNQLWTFRTDGTIRWLGKCLTASSSVMIYDCNTVPPEATKWVVSIDGTITNPHSGLVLTAPQAAEGTALSLENNIHAARQGWTVGDVEPLVTFIVGYKQMCLRENGENNFVWLEDCVLNRVQQEWALYGDGTIRVNSNRSLCVTSEDHEPSDLIVILKCEGSGNQRWVFNTNGTISNPNAKLLMDVAQRDVSLRKIILYRPTGNPNQQWI TTTHPA

TABLE 2 Sambucus Nigra Lectin 1′ (Genbank Accession No. U66191):(SEQ ID NO: 25) MKVVATILYLVVLAICGLGIHGAHPTHSAPPTVYPSVSFNLTEANSNEYRHFLQELRGKVILGSHRAFDLPVLNPESKVSDSDRFVLVRLTNPSRKKVTLAIDVVTFYVVAFAQNDRSYFFSGSSEVQRENLFVDTTQEDLNFKGDYTSLEHQVGFGRVYIPLGPKSLAQSISSLSTYKSSAGDNKRLARSLLVVIQMVSEAARFRYIQLRIQASITDAKEFTPDLLMLSMENKWSSMSSEIQQAQPGGAFAQVVKLLDQRNHPIDVTNFRRLFQLTSVAVLLHGCPTVTKMPAYIIKMPVFNGGEDEERCSVVEEVTRRIGGRDGFCAEVKNGDEKDGTPVQLSSCGEQSNQQWTFSTDGTIQSLGKCLTTSSSVMIYNCKVVPPESTKWVVSIDGTITNPRSGLVLTAPKAAEGTLVSLEKNVHAARQGWIVGNVEPLVTFIVGYEQMCLETNPGNNDVSLGDCSVKSASKVDQKWALYGDGTIRVNNDRSLCVTSEGKSSNEPIIILKCLGWANQRWVFNTDGTISNPDSKLVMHVDQNDVPLRKIILS HPSGTSNQQWIASTHPA

TABLE 3 Polyporous squamosus lectin la (UniProt Q75WT9) (SEQ ID NO: 26)MSFQGHGIYYIASAYVANTRLALSEDSSANKSPDVIISSDAVDPLNNLWLIEPVGEADTYTVRNAFAGSYMDLAGHAATDGTAIIGYRPTGGDNQKWIISQINDVWKIKSKETGTFVTLLNGDGGGTGTVVGWQNITNNTSQNWTFQKLSQTGANVHATLLACPALRQDFKSYLSDGLYLVLTRDQISSIWQASGLGSTPWRSEIFDCDDFATVFKGAVAKWGNENFKANGFALLCGLMFGSKSSGAHAYNWFVERGNFSTVTFFEPQNGTYSANAWDYKAY FGLF

TABLE 4 Polyporous squamosus lectin 1b (UniProt Q75WT8) (SEQ ID NO: 27)MSFEGHGIYHIPHAHVANIRMALANRGSGQNGTPVIAWDSNNDAFDHMWLVEPTGEADTYTIHNVSTGTYMDVTASAVADNTPIIGYQRTGNDNQKWIIRQVQTDGGDRPWKIQCKATGTFATLYSGGGSGTAIVGWRLVNSNGNQDWVFQKLSQTSVNVHATLLACGATVGQDFKNYLYDGLYLVLPRDRISAIWKASGLGETARRDGIYDSDEFAMTFKSAAATWGKENFKADGFAILCGMMFGTKASTNRHAYNWVVERGSFSTVTFFEPQNGTYSDDA WGYKAYFGLFAptamers

In some embodiments, binding agents provided in accordance with thepresent invention are aptamers. Aptamers are macromolecules composed ofnucleic acid (e.g., RNA, DNA) that bind tightly to a specific moleculartarget (e.g., an umbrella topology glycan). A particular aptamer may bedescribed by a linear nucleotide sequence and is typically about 15 toabout 60 nucleotides in length. Without wishing to be bound by anytheory, it is contemplated that the chain of nucleotides in an aptamerform intramolecular interactions that fold the molecule into a complexthree-dimensional shape, and this three-dimensional shape allows theaptamer to bind tightly to the surface of its target molecule. Given theextraordinary diversity of molecular shapes that exist within theuniverse of all possible nucleotide sequences, aptamers may be obtainedfor a wide array of molecular targets, including proteins and smallmolecules. In addition to high specificity, aptamers have very highaffinities for their targets (e.g., affinities in the picomolar to lownanomolar range for proteins). Aptamers are chemically stable and can beboiled or frozen without loss of activity. Because they are syntheticmolecules, they are amenable to a variety of modifications, which canoptimize their function for particular applications. For example,aptamers can be modified to dramatically reduce their sensitivity todegradation by enzymes in the blood for use in in vivo applications. Inaddition, aptamers can be modified to alter their biodistribution orplasma residence time.

Selection of aptamers that can bind umbrella topology glycans (and/or toumbrella topology glycan mimics) can be achieved through methods knownin the art. For example, aptamers can be selected using the SELEX(Systematic Evolution of Ligands by Exponential Enrichment) method(Tuerk, C., and Gold, L., 1990 Science 249:505; incorporated herein byreference). In the SELEX method, a large library of nucleic acidmolecules (e.g., 10¹⁵ different molecules) is produced and/or screenedwith the target molecule (e.g., an umbrella topology glycan of umbrellatopology glycan epitope). The target molecule is allowed to incubatewith the library of nucleotide sequences for a period of time. Severalmethods, known in the art, can then be used to physically isolate theaptamer target molecules from the unbound molecules in the mixture,which can be discarded. The aptamers with the highest affinity for thetarget molecule can then be purified away from the target molecule andamplified enzymatically to produce a new library of molecules that issubstantially enriched for aptamers that can bind the target molecule.The enriched library can then be used to initiate a new cycle ofselection, partitioning, and amplification. After 5-15 cycles of thisiterative selection, partitioning and amplification process, the libraryis reduced to a small number of aptamers that bind tightly to the targetmolecule. Individual molecules in the mixture can then be isolated,their nucleotide sequences determined, and their properties with respectto binding affinity and specificity measured and compared. Isolatedaptamers can then be further refined to eliminate any nucleotides thatdo not contribute to target binding and/or aptamer structure, therebyproducing aptamers truncated to their core binding domain. See Jayasena,S. D. 1999 Clin. Chem. 45:1628-1650, for review of aptamer technology;the entire teachings of which are incorporated herein by reference).

Production of Polypeptides

Inventive polypeptides (e.g., HA polypeptides and/or Non-HApolypeptides), and/or characteristic portions thereof, or nucleic acidsencoding them, may be produced by any available means.

Inventive polypeptides (or characteristic portions) may be produced, forexample, by utilizing a host cell system engineered to express aninventive polypeptide-encoding nucleic acid.

Any system can be used to produce polypeptides (or characteristicportions), such as egg, baculovirus, plant, yeast, Madin-Darby CanineKidney cells (MDCK), or Vero (African green monkey kidney) cells.Alternatively or additionally, polypeptides (or characteristic portions)can be expressed in cells using recombinant techniques, such as throughthe use of an expression vector (Sambrook et al., 1989 MolecularCloning: A Laboratory Manual, CSHL Press; incorporated herein byreference).

Alternatively or additionally, inventive polypeptides (or characteristicportions thereof) can be produced by synthetic means.

Alternatively or additionally, inventive polypeptides (or characteristicportions thereof), and particularly HA polypeptides, may be produced inthe context of intact virus, whether otherwise wild type, attenuated,killed, etc. Inventive polypeptides (e.g., HA polypeptides), orcharacteristic portions thereof, may also be produced in the context ofvirus like particles.

In some embodiments, HA polypeptides (or characteristic portionsthereof) can be isolated and/or purified from influenza virus. Forexample, virus may be grown in eggs, such as embryonated hen eggs, inwhich case the harvested material is typically allantoic fluid.Alternatively or additionally, influenza virus may be derived from anymethod using tissue culture to grow the virus. Suitable cell substratesfor growing the virus include, for example, dog kidney cells such asMDCK or cells from a clone of MDCK, MDCK-like cells, monkey kidney cellssuch as AGMK cells including Vero cells, cultured epithelial cells ascontinuous cell lines, 293T cells, BK-21 cells, CV-1 cells, or any othermammalian cell type suitable for the production of influenza virus forvaccine purposes, readily available from commercial sources (e.g., ATCC,Rockville, Md.). Suitable cell substrates also include human cells suchas MRC-5 cells. Suitable cell substrates are not limited to cell lines;for example primary cells such as chicken embryo fibroblasts are alsoincluded.

Also, it will be appreciated by those of ordinary skill in the art thatpolypeptides, and particularly variant HA polypeptides as describedherein, may be generated, identified, isolated, and/or produced byculturing cells or organisms that produce the polypeptide (whether aloneor as part of a complex, including as part of a virus particle orvirus), under conditions that allow ready screening and/or selection ofpolypeptides capable of binding to umbrella-topology glycans. To givebut one example, in some embodiments, it may be useful to produce and/orstudy a collection of polypeptides (e.g., HA variant polypeptides) underconditions that reveal and/or favor those variants that bind to umbrellatopology glycans (e.g., with particular specificity and/or affinity). Insome embodiments, such a collection of polypeptides (e.g., HA variantpolypeptides) results from evolution in nature. In some embodiments,such a collection of polypeptides (e.g., HA variant polypeptides)results from engineering. In some embodiments, such a collection ofpolypeptides (e.g., HA variant polypeptides) results from a combinationof engineering and natural evolution.

HA Receptors

HA interacts with the surface of cells by binding to a glycoproteinreceptor. Binding of HA to HA receptors is predominantly mediated byN-linked glycans on the HA receptors. Specifically, HA on the surface offlu virus particles recognizes sialylated glycans that are associatedwith HA receptors on the surface of the cellular host. After recognitionand binding, the host cell engulfs the viral cell and the virus is ableto replicate and produce many more virus particles to be distributed toneighboring cells. Some crystal structures of exemplary HA-glycaninteractions have been identified and are presented in Table 5:

TABLE 5 Crystal Structures of HA-Glycan Complexes Abbreviation (PDB ID)Virus Strain Glycan (with assigned coordinates) ADkALB76_H1_26 (2WRH)A/duck/Alberta/76 (H1N1) Neu5Ac ASI30_H1_23 (1RV0)A/Swine/Iowa/30 (H1N1) Neu5Ac ASI30_H1_26 (1RVT) A/Swine/Iowa/30 (H1N1)Neu5AcαGalβ4GlcNAcβ3Galβ4Glc ASC18_H1_26 (2WRG)A/South Carolina/1/18 (H1N1) Neu5Acα6Galβ4GlcNAcβ3Gal APR34_H1_23 (1RVX)A/Puerto Rico/8/34 (H1N1) Neu5Acα3Galβ4GlcNAc APR34_H1_26 (1RVZ)A/Puerto Rico/8/34 (H1N1) Neu5Acα6Galβ4GlcNAc ACkNY91_H2_23 (2WR2)A/chicken/NY/29878/91 (H2N2) Neu5Acα3Galβ3GlcNAc ACkNY91_H2_26 (2WR1)A/chicken/NY/29878/91 (H2N2) Neu5Acα6Galβ4GlcNAc ADkON77_H2_23 (2WR3)A/duck/Ontario/77 (H2N2) Neu5Acα3Galβ4GlcNAc ADkON77_H2_26 (2WR4)A/duck/Ontario/77 (H2N2) Neu5Acα6GalβGlcNAc ACkPD84_H2_26 (2WRF)A/chicken/Potsdam/475/84 (H2N2) Neu5Acα6Gal ASING57_H2_23 (2WRB)A/Singapore/1/57 (H2N2) Neu5Ac ASING57_H2_26 (2WR7)A/Singapore/1/57 (H2N2) Neu5Acα6Galβ4GlcNAcβ3Gal AJAP57_H2_26 (2WRE)A/Japan/305/57 (H2N2) Neu5Acα6Gal ADU63_H3_23 (1MQM)A/Duck/Ukraine/1/63 (H3N8) Neu5Acα3Gal ADU63_H3_26 (1MQN)A/Duck/Ukraine/1/63 (H3N8) Neu5Acα6Gal AAI68_H3_23 (1HGG)A/Aichi/2/68 (H3N2) Neu5Acα3Galβ4Glc ADS97_H5_23 (1JSN)A/Duck/Singapore/3/97 (H5N3) Neu5Acα3Galβ3GlcNAc ADS97_H5_26 (1JSO)A/Duck/Singapore/3/97 (H5N3) Neu5Ac Viet04_H5 (2FK0)A/Vietnam/1203/2004 (H5N1) HA-α2-6 sialylated glycan complexes weregenerated by superimposition of the CA trace of the HA1 subunit ofADU63_H3 and ADS97_H5 and Viet04_H5 on ASI30_H1_26 and APR34_H1_26 (H1).Although the structural complexes of the human A/Aichi/2/68 (H3N2) withα2-6 sialylated glycans are published (Eisen et al., 1997, Virology,232: 19), their coordinates were not available in the Protein Data Bank.The SARF2 (http://123d.ncifcrf/gov/sarf2.html) program was used toobtain the structural alignment of the different HA1 subunits forsuperimposition.

HA receptors are modified by either α2-3 or α2-6 sialylated glycans nearthe receptor's HA-binding site, and the type of linkage of thereceptor-bound glycan can affect the conformation of the receptor'sHA-binding site, thus affecting the receptor's specificity for differentHAs.

For example, the glycan binding pocket of avian HA is narrow. Accordingto the present invention, this pocket binds to the trans conformation ofα2-3 sialylated glycans, and/or to cone-topology glycans, whether α2-3or α2-6 linked.

HA receptors in avian tissues, and also in human deep lung andgastrointestinal (GI) tract tissues are characterized by α2-3 sialylatedglycan linkages, and furthermore (according to the present invention),are characterized by glycans, including α2-3 sialylated and/or α2-6sialylated glycans, which predominantly adopt cone topologies. HAreceptors having such cone-topology glycans may be referred to herein asCTHArs.

By contrast, human HA receptors in the bronchus and trachea of the upperrespiratory tract are modified by α2-6 sialylated glycans. Unlike theα2-3 motif, the α2-6 motif has an additional degree of conformationalfreedom due to the C6-C5 bond (Russell et al., 2006 Glycoconj J 23:85;incorporated herein by reference). HAs that bind to such α2-6 sialylatedglycans have a more open binding pocket to accommodate the diversity ofstructures arising from this conformational freedom. Moreover, accordingto the present invention, HAs may need to bind to glycans (e.g., α2-6sialylated glycans) in an umbrella topology, and particularly may needto bind to such umbrella topology glycans with strong affinity and/orspecificity, in order to effectively mediate infection of human upperrespiratory tract tissues. HA receptors having umbrella-topology glycansmay be referred to herein as UTHArs.

As a result of these spatially restricted glycosylation profiles, humansare not usually infected by viruses containing many wild type avian HAs(e.g., avian H2). Specifically, because the portions of the humanrespiratory tract that are most likely to encounter virus (i.e., thetrachea and bronchi) lack receptors with cone glycans (e.g., α2-3sialylated glycans, and/or short glycans) and wild type avian HAstypically bind primarily or exclusively to receptors associated withcone glycans (e.g., α2-3 sialylated glycans, and/or short glycans),humans rarely become infected with avian viruses. Only when insufficiently close contact with virus that it can access the deep lungand/or gastrointestinal tract receptors having umbrella glycans (e.g.,long α2-6 sialylated glycans) do humans become infected.

Glycan Arrays

To rapidly expand the current knowledge of known specific glycan-glycanbinding protein (GBP) interactions, the Consortium for FunctionalGlycomics (CFG; www.functionalglycomics.org), an internationalcollaborative research initiative, has developed glycan arrayscomprising several glycan structures that have enabled high throughputscreening of GBPs for novel glycan ligand specificities. The glycanarrays comprise both monovalent and polyvalent glycan motifs (i.e.attached to polyacrylamide backbone), and each array comprises 264glycans with low (10 μM) and high (100 μM) concentrations, and six spotsfor each concentration (seehttp://www.functionalglycomics.org/static/consortium/resources/resourcecoreh5.shtml).

The arrays predominantly comprise synthetic glycans that capture thephysiological diversity of N- and O-linked glycans. In addition to thesynthetic glycans, N-linked glycan mixtures derived from differentmammalian glycoproteins are also represented on the array.

As used herein, a glycan “array” refers to a set of one or more glycans,optionally immobilized on a solid support. In some embodiments, an“array” is a collection of glycans present as an organized arrangementor pattern at two or more locations that are physically separated inspace. Typically, a glycan array will have at least 4, at least 8, atleast 16, at least 24, at least 48, at least 96 or several hundred orthousand discrete locations. In general, inventive glycan arrays mayhave any of a variety of formats. Various different array formatsapplicable to biomolecules are known in the art. For example, a hugenumber of protein and/or nucleic acid arrays are well known. Those ofordinary skill in the art will immediately appreciate standard arrayformats appropriate for glycan arrays of the present invention.

In some embodiments, inventive glycan arrays are present in “microarray”formats. A microarray may typically have sample locations separated by adistance of about 50 to about 200 microns or less and immobilized samplein the nano to micromolar range or nano to picogram range. Array formatsknown in the art include, for example, those in which each discretesample location has a scale of, for example, ten microns.

In some embodiments, inventive glycan arrays comprise a plurality ofglycans spatially immobilized on a support. The present inventionprovides glycan molecules arrayed on a support. As used herein,“support” refers to any material which is suitable to be used to arrayglycan molecules. As will be appreciated by those of ordinary skill inthe art, any of a wide variety of materials may be employed. To give buta few examples, support materials which may be of use in the inventioninclude hydrophobic membranes, for example, nitrocellulose, PVDF ornylon membranes. Such membranes are well known in the art and can beobtained from, for example, Bio-Rad, Hemel Hempstead, UK.

In some embodiments, the support on which glycans are arrayed maycomprise a metal oxide. Suitable metal oxides include, but are notlimited to, titanium oxide, tantalum oxide, and aluminum oxide. Examplesof such materials may be obtained from Sigma-Aldrich Company Ltd, FancyRoad, Poole, Dorset. BH12 4QH UK.

In some embodiments, such a support is or comprises a metal oxide gel. Ametal oxide gel is considered to provide a large surface area within agiven macroscopic area to aid immobilization of thecarbohydrate-containing molecules.

Additional or alternative support materials which may be used inaccordance with the present invention include gels, for example silicagels or aluminum oxide gels. Examples of such materials may be obtainedfrom, for example, Merck KGaA, Darmstadt, Germany.

In some embodiments, glycan arrays are immobilized on a support that canresist change in size or shape during normal use. For example a supportmay be a glass slide coated with a component material suitable to beused to array glycans. Also, some composite materials can desirableprovide solidity to a support.

As demonstrated herein, inventive arrays are useful for theidentification and/or characterization of different HA polypeptides andtheir binding characteristics. In some embodiments, HA polypeptides inaccordance with the invention are tested on such arrays to assess theirability to bind to umbrella topology glycans (e.g., to α2-6 sialylatedglycans, and particularly to long α2-6 sialylated glycans arranged in anumbrella topology).

Indeed, the present invention provides arrays of α2-6 sialylatedglycans, and optionally α2-3 sialylated glycans, that can be used tocharacterize HA polypeptide binding capabilities and/or as a diagnosticto detect, for example, human-binding HA polypeptides. In someembodiments, inventive arrays contain glycans (e.g., α2-6 sialylatedglycans, and particularly long α2-6 sialylated glycans) in an umbrellatopology. As will be clear to those of ordinary skill in the art, sucharrays are useful for characterizing or detecting any HA polypeptides,including for example, those found in natural influenza isolates inaddition to those designed and/or prepared by researchers.

In some embodiments, such arrays include glycans representative of about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90% about 95%, or more of the glycans(e.g., the umbrella glycans, which will often be α2-6 sialylatedglycans, particularly long α2-6 sialylated glycans) found on human HAreceptors, and particularly on human upper respiratory tract HAreceptors. In some embodiments, inventive arrays include some or all ofthe glycan structures depicted in FIG. 14 In some embodiments, arraysinclude at least about 10%, about 15%, about 20%, about 25%, about 30%about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90% about 95%, ormore of these depicted glycans.

The present invention provides methods for identifying or characterizingHA polypeptides using glycan arrays. In some embodiments, for example,such methods comprise steps of (1) providing a sample containing HApolypeptide, (2) contacting the sample with a glycan array comprising,and (3) detecting binding of HA polypeptide to one or more glycans onthe array.

Suitable sources for samples containing HA polypeptides to be contactedwith glycan arrays according to the present invention include, but arenot limited to, pathological samples, such as blood, serum/plasma,peripheral blood mononuclear cells/peripheral blood lymphocytes(PBMC/PBL), sputum, urine, feces, throat swabs, dermal lesion swabs,cerebrospinal fluids, cervical smears, pus samples, food matrices, andtissues from various parts of the body such as brain, spleen, and liver.Alternatively or additionally, other suitable sources for samplescontaining HA polypeptides include, but are not limited to,environmental samples such as soil, water, and flora. Yet other samplesinclude laboratory samples, for example of engineered HA polypeptidesdesigned and/or prepared by researchers. Other samples that have notbeen listed may also be applicable.

A wide variety of detection systems suitable for assaying HA polypeptidebinding to inventive glycan arrays are known in the art. For example, HApolypeptides can be detectably labeled (directly or indirectly) prior toor after being contacted with the array; binding can then be detected bydetection of localized label. In some embodiments, scanning devices canbe utilized to examine particular locations on an array.

Alternatively or additionally, binding to arrayed glycans can bemeasured using, for example, calorimetric, fluorescence, or radioactivedetection systems, or other labeling methods, or other methods that donot require labeling. In general, fluorescent detection typicallyinvolves directly probing the array with a fluorescent molecule andmonitoring fluorescent signals. Alternatively or additionally, arrayscan be probed with a molecule that is tagged (for example, with biotin)for indirect fluorescence detection (in this case, by testing forbinding of fluorescently-labeled streptavidin). Alternatively oradditionally, fluorescence quenching methods can be utilized in whichthe arrayed glycans are fluorescently labeled and probed with a testmolecule (which may or may not be labeled with a different fluorophore).In such embodiments, binding to the array acts to squelch thefluorescence emitted from the arrayed glycan, therefore binding isdetected by loss of fluorescent emission. Alternatively or additionally,arrayed glycans can be probed with a live tissue sample that has beengrown in the presence of a radioactive substance, yielding aradioactively labeled probe. Binding in such embodiments can be detectedby measuring radioactive emission.

Such methods are useful to determine the fact of binding and/or theextent of binding by HA polypeptides to inventive glycan arrays. In someembodiments of the invention, such methods can further be used toidentify and/or characterize agents that interfere with or otherwisealter glycan-HA polypeptide interactions.

Methods described below may be of particular use in, for example,identifying whether a molecule thought to be capable of interacting witha carbohydrate can actually do so, or to identify whether a moleculeunexpectedly has the capability of interacting with a carbohydrate.

The present invention also provides methods of using inventive arrays,for example, to detect a particular agent in a test sample. Forinstance, such methods may comprise steps of (1) contacting a glycanarray with a test sample (e.g., with a sample thought to contain an HApolypeptide); and, (2) detecting the binding of any agent in the testsample to the array.

Yet further, binding to inventive arrays may be utilized, for example,to determine kinetics of interaction between binding agent and glycan.For example, inventive methods for determining interaction kinetics mayinclude steps of (1) contacting a glycan array with the molecule beingtested; and, (2) measuring kinetics of interaction between the bindingagent and arrayed glycan(s).

The kinetics of interaction of a binding agent with any of the glycansin an inventive array can be measured by real time changes in, forexample, colorimetric or fluorescent signals, as detailed above. Suchmethods may be of particular use in, for example, determining whether aparticular binding agent is able to interact with a specificcarbohydrate with a higher degree of binding than does a differentbinding agent interacting with the same carbohydrate.

It will be appreciated, of course, that glycan binding by HApolypeptides in accordance with the invention can be evaluated on glycansamples or sources not present in an array format per se. For example,HA polypeptides in accordance with the invention can be bound to tissuesamples and/or cell lines to assess their glycan bindingcharacteristics. Appropriate cell lines include, for example, any of avariety of mammalian cell lines, particularly those expressing HAreceptors containing umbrella topology glycans (e.g., at least some ofwhich may be α2-6 sialylated glycans, and particularly long α2-6sialylated glycans). In some embodiments, utilized cell lines expressindividual glycans with umbrella topology. In some embodiments, utilizedcell lines express a diversity of glycans. In some embodiments, celllines are obtained from clinical isolates; in some they are maintainedor manipulated to have a desired glycan distribution and/or prevalence.In some embodiments, tissue samples and/or cell lines express glycanscharacteristic of mammalian upper respiratory epithelial cells.

Data Mining Platform

As discussed here, according to the present invention, HA polypeptidescan be identified and/or characterized by mining data from glycanbinding studies, structural information (e.g., HA crystal structures),and/or protein structure prediction programs.

The main steps involved in the particular data mining process utilizedby the present inventors (and exemplified herein) are illustrated inFIG. 15. These steps involved operations on three elements: dataobjects, features, and classifiers. “Data objects” were the raw datathat were stored in a database. In the case of glycan array data, thechemical description of glycan structures in terms of monosaccharidesand linkages and their binding signals with different GBPs screenedconstituted the data objects. Properties of the data objects were“features.” Rules or patterns obtained based on the features were chosento describe a data object. “Classifiers” were the rules or patterns thatwere used to either cluster data objects into specific classes ordetermine relationships between or among features. The classifiersprovided specific features that were satisfied by the glycans that bindwith high affinity to a GBP. These rules were of two kinds (1) featurespresent on a set of high affinity glycan ligands, which can beconsidered to enhance binding, and (2) features that should not bepresent in the high affinity glycan ligands, which can be considered notfavorable for binding.

The data mining platform utilized herein comprised software modules thatinteract with each other (FIG. 15) to perform the operations describedabove. The feature extractor interfaces to the CFG database to extractfeatures, and the object-based relational database used by CFGfacilitates the flexible definition of features.

Feature Extraction and Data Preparation

Representative features extracted from the glycans on the glycan arrayare listed in Table 6.

TABLE 6 Features extracted from the glycans on the glycan array.Features extracted Feature Description Monosaccharide level CompositionNumber of hex, hexNAcs, dHex, sialic acids, etc [In FIG. 1, thecomposition is Hex = 5; HexNAc = 4]. Terminal composition is distinctlyrecorded [In FIG. 1, the terminal composition is Hex = 2; HexNAc = 2].Explicit Composition Number of Glc, Gal, GlcNAc, Fuc, GalNAc, Neu5Ac,Neu5Gc, etc [In FIG. 1, the explicit composition is Man = 5; GlcNAc =4]. Terminal explicit composition is explicitly recorded [In FIG. 1, theterminal explicit composition is Man = 2; GlcNAc = 2]. Higher orderfeatures Pairs Pair refers to a pair of monosaccharide, connectedcovalently by a linkage. The pairs are classified into two categories,regular [B] and terminal [T] to distinguish between the pair with onemonosaccharide that terminates in the non reducing end [FIG. 16]. Thefrequency of the pairs were extracted as features Triplets Tripletrefers to a set of three monosaccharides connected covalently by twolinkages. We classify them into three categories namely regular [B],terminal [T] and surface [S] [FIG. 16]. The compositions of eachcategory of triplets were extracted as features Quadruplets Similar tothe triplet features, quadruplets features are also extracted, with fourmonosaccharides and their linkages [FIG. 16]. Quadruplets are classifiedinto two varieties regular [B] and surface [S]. The frequencies of thedifferent quadruplets were extracted as features Clusters In the case ofsurface triplets and quadruplets above, if the linkage information isignored, we get a set of monosaccharide clusters, and their frequency ofoccurrence (composition) is tabulated. These features were chosen toanalyze the importance of types of linkages between the monosaccharides.Average Leaf Depth As an indicator of the effective length of theprobes, average depth of the reducing end of the tree is extracted as aglycan feature. In FIG. 16B, the leaf depths are 3, 4 and 3, and theaverage is 3.34 Number of Leaves As a measure of spread of the glycantree, the number of non reducing monosaccharides is extracted as afeature. For FIG. 16B, the number of leaves is 3. For FIG. 1 it is 4.GBP binding features These features are obtained for all GBPs screenedusing the array Mean signal per glycan Raw signal value averaged overtriplicate or quadruplicate [depending on array version] representationof the same glycan Signal to Noise Ratio Mean noise computed based onnegative control [standardized method developed by CFG] to calculatesignal to noise ratio [S/N] The features described in this table wereused by the rule based classification algorithm to identify patternsthat characterized binding to specific GBP.

The rationale behind choosing these particular features shown was thatglycan binding sites on GBPs typically accommodate di-tetra-saccharides.A tree based representation was used to capture the information onmonosaccharides and linkages in the glycan structures (root of the treeat the reducing end). This representation facilitated the abstraction ofvarious features including higher order features such as connected setof monosaccharide triplets, etc (FIG. 16). The data preparation involvedgenerating a column-wise listing of all glycans in the glycan arrayalong with abstracted features (Table 6) for each glycan. From thismaster table of glycans and their features, a subset is chosen for therule based classification (see below) to determine specific patternsthat govern the binding to a specific GBP or set of GBPs.

Classifiers

Different types of classifiers have been developed and used in manyapplications. They fall primarily into three main categories:Mathematical Methods, Distance Methods and Logic Methods. Thesedifferent methods and their advantages and disadvantages are discussedin detail in Weiss & Indrukhya (Predictive data mining—A practicalguide. Morgan Kaufmann, San Francisco, 1998). For this specificapplication we chose a method called Rule Induction, which falls underLogic Methods. The Rule Induction classifier generates patterns in formof IF-THEN rules.

One of the main advantages of the Logic Methods, and specificallyclassifiers such as the Rule Induction method that generate IF-THENrules, is that the results of the classifiers can be explained moreeasily when compared to the other statistical or mathematical methods.This allows one to explore the structural and biological significance ofthe rule or pattern discovered. An example rule generated using thefeatures described earlier (Table 6) is: IF A Glycan contains“Galb4GlcNAcb3Gal[B]” and DOES NOT contain “Fuca3GlcNAc[B]”, THEN theGlycan will bind with higher affinity to Galectin 3. The specific RuleInduction algorithm that was used in this case is the one developed byWeiss & Indurkya (Predictive data mining—A practical guide. MorganKaufmann, San Francisco, 1998.

Binding Levels

A threshold that distinguished low affinity and high affinity bindingwas defined for each of the glycan array screening data sets.

Nucleic Acids

In some embodiments, the present invention provides nucleic acids whichencode an HA polypeptide or a characteristic or biologically activeportion of an HA polypeptide. In other embodiments, the inventionprovides nucleic acids which are complementary to nucleic acids whichencode an HA polypeptide or a characteristic or biologically activeportion of an HA polypeptide.

In some embodiments, the invention provides nucleic acid molecules whichhybridize to nucleic acids encoding an HA polypeptide or acharacteristic or biologically active portion of an HA polypeptide. Suchnucleic acids can be used, for example, as primers or as probes. To givebut a few examples, such nucleic acids can be used as primers inpolymerase chain reaction (PCR), as probes for hybridization (includingin situ hybridization), and/or as primers for reverse transcription-PCR(RT-PCR).

In some embodiments, nucleic acids can be DNA or RNA, and can be singlestranded or double-stranded. In some embodiments, inventive nucleicacids may include one or more non-natural nucleotides; in otherembodiments, nucleic acids in accordance with the present inventioninclude only natural nucleotides.

Antibodies to Polypeptides

The present invention provides antibodies to binding agent polypeptidesin accordance with the present invention (e.g., HA polypeptides). Thesemay be monoclonal or polyclonal and may be prepared by any of a varietyof techniques known to those of ordinary skill in the art (e.g., seeHarlow and Lane, 1988 Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory; incorporated herein by reference). For example,antibodies can be produced by cell culture techniques, including thegeneration of monoclonal antibodies, or via transfection of antibodygenes into suitable bacterial or mammalian cell hosts, in order to allowfor the production of recombinant antibodies.

Testing Binding Agents in Animal Models

The present invention provides methods for testing binding agents inaccordance with the present invention (e.g., HA polypeptides, LSBAs,USBAs, UTSBAs, etc.) in an animal host. As used herein, an “animal host”includes any animal model suitable for influenza research. For example,animal hosts suitable for the invention can be any mammalian hosts,including primates, ferrets, cats, dogs, cows, horses, rodents such as,mice, hamsters, rabbits, and rats. In some embodiments, an animal hostused for the invention is a ferret. In particular, in some embodiments,an animal host is naïve to viral exposure or infection prior toadministration of an inventive binding agent (optionally in an inventivecomposition). In some embodiments, the animal host is inoculated with,infected with, or otherwise exposed to virus prior to or concurrent withadministration of an inventive binding agent. An animal host used in thepractice of the present invention can be inoculated with, infected with,or otherwise exposed to virus by any method known in the art. In someembodiments, an animal host may be inoculated with, infected with, orexposed to virus intranasally.

In some embodiments, a suitable animal host may have a similardistribution of umbrella vs. cone topology glycans and/or α2-6 glycansvs. α2-3 glycans to the distribution found in the human respiratorytract. For example, it is contemplated that a ferret as an animal hostmay be more representative than a mouse when used as model of diseasecaused by influenza viruses in humans (Tumpey, et al. 2007 Science 315;655-659; incorporated herein by reference). Without wishing to be boundany theories, the present invention encompasses the idea that ferretsmay have a more similar distribution of glycans in the respiratory tractto those in the human respiratory tract than mouse does to human.

Naïve and/or inoculated animals may be used for any of a variety ofstudies. For example, such animal models may be used for virustransmission studies as in known in the art. It is contemplated that theuse of ferrets in virus transmission studies may serve as a reliablepredictor for virus transmission in humans. For example, airtransmission of viral influenza from inoculated animals (e.g., ferrets)to naïve animals is known in the art (Tumpey, et al. 2007 Science 315;655-659; incorporated herein by reference). Virus transmission studiesmay be used to test inventive binding agent polypeptides (e.g., HApolypeptides). For example, inventive binding agents may be administeredto a suitable animal host before, during or after virus transmissionstudies in order to determine the efficacy of said binding agent inblocking virus binding and/or infectivity in the animal host. Usinginformation gathered from virus transmission studies in an animal host,one may predict the efficacy of a binding agent in blocking virusbinding and/or infectivity in a human host.

Treatment

The present invention provides systems, compositions, and methods totreat (e.g., alleviate, ameliorate, relieve, delay onset of, inhibitprogression of, reduce severity of, and/or reduce incidence of one ormore symptoms or features of) and/or prevent influenza infection. Insome embodiments, inventive binding agents such as those describedherein may be used for a variety of therapeutic purposes, e.g., treatinginfluenza infection and/or developing vaccines to immunize subjectsagainst influenza infection.

A. Vaccination

In some embodiments, inventive binding agents in accordance with theinvention (e.g., entities that bind to HA polypeptides and/or fragments,variants, and/or characteristic portions thereof; entities that bind toumbrella-topology glycans) may be utilized for prophylacticapplications. In some embodiments, prophylactic applications involvesystems and methods for preventing, inhibiting progression of, and/ordelaying the onset of influenza infection.

In some embodiments, influenza vaccines are used to prevent and/or delayonset of infection by influenza. In some embodiments, vaccination istailored to a particular HA polypeptide. For example, vaccinecompositions may comprise H2 HA polypeptides and/or variants, fragments,and/or characteristic portions thereof. In some embodiments, it isdesirable for vaccine compositions to comprise antigens that have anative conformation, mediate a protective response (e.g., complementactivation, virus neutralization, etc.), and/or can induce a strongantibody response.

In some embodiments, interfering agents may be utilized for passiveimmunization (i.e., immunization wherein antibodies are administered toa subject). In some embodiments, influenza vaccines for passiveimmunization may comprise antibody interfering agents, such as thosedescribed herein. In some embodiments, passive immunization occurs whenantibodies are transferred from mother to fetus during pregnancy. Insome embodiments, antibodies are administered directly to an individual(e.g., by injection, orally, etc.).

The present invention provides influenza vaccines for activeimmunization (i.e., immunization wherein microbes, proteins, peptides,epitopes, mimotopes, etc. are administered to a subject). In someembodiments, influenza vaccines may comprise one or more interferingagents and/or binding agents, as described herein.

In some embodiments, vaccines comprise at least one HA polypeptide(and/or to variants, fragments, and/or characteristic portions thereof),e.g., any of the HA polypeptides, variants, fragments, characteristicportions, and/or combinations thereof described herein. In someembodiments, vaccines comprise H2 HA polypeptides (and/or to variants,fragments, and/or characteristic portions thereof). In some embodiments,vaccines comprise HA polypeptides having one or more of the following:arginine at Residue 137, threonine at Residue 193, leucine at Residue226, and/or serine at Residue 228. In some embodiments, vaccinescomprise HA polypeptides having each of the following: arginine atResidue 137, threonine at Residue 193, leucine at Residue 226, and/orserine at Residue 228. In some embodiments, vaccines comprise liveactive virus particles comprising one or more of any HA polypeptidedescribed herein, live attenuated virus particles comprising one or moreof any HA polypeptide described herein, virus-like particles (VLPs)comprising one or more of any HA polypeptide described herein, subunitvaccines comprising one or more of any HA polypeptide described herein,and/or combinations thereof.

In some embodiments, a vaccine composition comprises at least oneadjuvant. Any adjuvant may be used in accordance with the presentinvention. A large number of adjuvants are known; a useful compendium ofmany such compounds is prepared by the National Institutes of Health andcan be found on the internet(www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf). See also Allison(1998, Dev. Biol. Stand., 92:3-11; incorporated herein by reference),Unkeless et al. (1998, Annu. Rev. Immunol., 6:251-281; incorporatedherein by reference), and Phillips et al. (1992, Vaccine, 10:151-158;incorporated herein by reference). Hundreds of different adjuvants areknown in the art and could be employed in the practice of the presentinvention. Exemplary adjuvants that can be utilized in accordance withthe invention include, but are not limited to, cytokines, aluminum salts(e.g., aluminum hydroxide, aluminum phosphate, etc.; Baylor et al.,Vaccine, 20:S18, 2002; incorporated herein by reference), gel-typeadjuvants (e.g., calcium phosphate, etc.); microbial adjuvants (e.g.,immunomodulatory DNA sequences that include CpG motifs; endotoxins suchas monophosphoryl lipid A (Ribi et al., 1986, Immunology andImmunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY, p407, 1986; incorporated herein by reference); exotoxins such as choleratoxin, E. coli heat labile toxin, and pertussis toxin; muramyldipeptide, etc.); oil-emulsion and emulsifier-based adjuvants (e.g.,Freund's Adjuvant, MF59 [Novartis], SAF, etc.); particulate adjuvants(e.g., liposomes, biodegradable microspheres, etc.); synthetic adjuvants(e.g., nonionic block copolymers, muramyl peptide analogues,polyphosphazene, synthetic polynucleotides, etc.); and/or combinationsthereof. Other exemplary adjuvants include some polymers (e.g.,polyphosphazenes; described in U.S. Pat. No. 5,500,161, which isincorporated herein by reference), Q57, saponins (e.g., QS21, Ghochikyanet al., Vaccine, 24:2275, 2006; incorporated herein by reference),squalene, tetrachlorodecaoxide, CPG 7909 (Cooper et al., Vaccine,22:3136, 2004; incorporated herein by reference),poly[di(carboxylatophenoxy)phosphazene] (PCCP; Payne et al., Vaccine,16:92, 1998; incorporated herein by reference), interferon-γ (Cao etal., Vaccine, 10:238, 1992; incorporated herein by reference), blockcopolymer P1205 (CRL1005; Katz et al., Vaccine, 18:2177, 2000;incorporated herein by reference), interleukin-2 (IL-2; Mbwuike et al.,Vaccine, 8:347, 1990; incorporated herein by reference), polymethylmethacrylate (PMMA; Kreuter et al., J. Pharm. Sci., 70:367, 1981;incorporated herein by reference), etc.

B. Therapy

The present invention provides systems and methods for treating patientssuffering from, susceptible to, and/or displaying symptoms of influenzainfection. In some embodiments, the invention provides systems andmethods useful for stratifying patients suffering from, susceptible to,and/or displaying symptoms of influenza infection.

In some embodiments, inventive binding agents in accordance with theinvention may be utilized for therapeutic applications.

In some embodiments, therapeutic applications comprise administering atherapeutically effective amount of at least one binding agent inaccordance with the invention to a subject in need thereof. In someembodiments, administration of binding agents to a subject mayalleviate, ameliorate, relieve, delay onset of, inhibit progression of,reduce severity of, and/or reduce incidence of one or more signs,symptoms, and/or features of influenza infection.

In some embodiments, administration of binding agents reduces the levelof influenza virions circulating in a subject (e.g., influenza virionsthat are capable of infecting new cells). In some embodiments,administration of binding agents reduces the level of influenza virionscirculating in a subject by about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about99%, or about 100% relative to non-treated controls.

In some embodiments, binding agents may be used in vitro to reduce viralload in a subject. For reducing viral load of a body component,particularly a body component of a patient infected with influenza, apatient's blood is passed through a device comprising binding agentsbound to a surface or solid support for capturing influenza virions(see, for example, U.S. Pat. Nos. 5,698,390 and 4,692,411; both of whichare incorporated herein by reference). Various other devices found inthe literature can be used with the subject antibodies to achieve asimilar result. A body component can be a biological fluid (e.g., blood,serum, etc.), a tissue, an organ, such as the liver, and the like.

In some embodiments, the “level of influenza virions circulating in asubject” refers to an absolute number of virions circulating in asubject. In some embodiments, the “level of influenza virionscirculating in a subject” refers to the number of virions per unitvolume (e.g., milliliter, liter, etc.) of the subject's blood. In someembodiments, the “level of influenza virions circulating in a subject”refers to viral load.

In some embodiments, administration of binding agents inhibits bindingof virus to HA receptors. In some embodiments, administration of bindingagents inhibits binding of virus to at least one HA receptor by about2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about50-fold, about 100-fold, about 500-fold, about 1000-fold, about10,000-fold, or greater than about 10,000-fold relative to non-treatedcontrols.

In some embodiments, administration of binding agents kills and/orinactivates influenza virions in a subject. In some embodiments,administration of influenza antibodies kills and/or inactivates about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, about 99%, or about 100% of influenzavirions in a subject relative to non-treated controls.

In some embodiments, administration of binding agents inhibitsvirus-mediated fusion with a target cell. In some embodiments,administration of binding agents inhibits virus-mediated fusion with atarget cell by about 2-fold, about 3-fold, about 4-fold, about 5-fold,about 10-fold, about 50-fold, about 100-fold, about 500-fold, about1000-fold, about 10,000-fold, or greater than about 10,000-fold relativeto non-treated controls.

In some embodiments, administration of binding agents inhibitsconformational changes of one or more proteins associated with virusentry. In some embodiments, administration of binding agents inhibitsconformational changes of one or more proteins associated with virusentry by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about10-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold,about 10,000-fold, or greater than about 10,000-fold relative tonon-treated controls.

In some embodiments, administration of binding agents results inconformational changes in HA polypeptides and/or HA receptors. Forexample, administered interfering agents and/or binding agents may bindto HA polypeptides and/or HA receptors, thereby sterically blocking theHA polypeptide's and/or HA receptors' ability to recognize and/orinteract with one another. In some embodiments, administered bindingagents may bind to HA polypeptides and/or HA receptors, thereby changingthe three-dimensional conformation of the HA polypeptides and/or HAreceptors in such a way that renders HA polypeptides and/or HA receptorsincapable of recognizing one another.

In some embodiments, treatment and/or vaccination regimens areparticularly tailored for the individual being treated and/orvaccinated. The present invention provides systems, compositions, andmethods useful for determining whether a patient is infected with H2 HAinfluenza or non-H2 HA influenza. Such methods can be utilized tostratify patients into treatment and/or vaccination categories. In someembodiments, such methods may be advantageous because the treatmentand/or vaccination is tailored to the particular individual beingtreated and/or vaccinated. To give but one particular example, if apatient is classified as being infected with H2 HA influenza, therapiesthat are useful for treatment of H2 HA influenza can be administered tothe patient, and therapies that are not useful for treatment of H2 HAinfluenza will not be administered. This avoids or reduces the risk ofadverse reactions from administering therapeutics that are not needed.Such methods eliminate the expense of treating and/or vaccinatingpatients who would not benefit from such treatment and/or vaccination.

C. Pharmaceutical Compositions

In some embodiments, the present invention provides for pharmaceuticalcompositions including inventive binding agents (e.g., HA polypeptides,LSBAs, UTBAs, UTBSAs, etc.) and/or related entities. For example, insome embodiments, binding agent polypeptide(s) (e.g., HA polypeptides),nucleic acids encoding such polypeptides, characteristic or biologicallyactive fragments of such polypeptides or nucleic acids, antibodies thatbind to and/or compete with such polypeptides or fragments, smallmolecules that interact with or compete with such polypeptides or withglycans that bind to them, etc. are included in inventive pharmaceuticalcompositions. In some embodiments, inventive binding agents that are notpolypeptides, e.g., that are small molecules, umbrella topology glycansand mimics thereof, carbohydrates, aptamers, polymers, nucleic acids,etc., are included in pharmaceutical compositions.

The invention encompasses treatment of influenza infections byadministration of such inventive pharmaceutical compositions. In someembodiments, inventive pharmaceutical compositions are administered to asubject suffering from or susceptible to an influenza infection. In someembodiments, a subject is considered to be suffering from an influenzainfection in the subject is displaying one or more symptoms commonlyassociated with influenza infection. In some embodiments, the subject isknown or believed to have been exposed to the influenza virus. In someembodiments, a subject is considered to be susceptible to an influenzainfection if the subject is known or believed to have been exposed tothe influenza virus. In some embodiments, a subject is known or believedto have been exposed to the influenza virus if the subject has been incontact with other individuals known or suspected to have been infectedwith the influenza virus and/or if the subject is or has been present ina location in which influenza infection is known or thought to beprevalent.

In some embodiments, subjects suffering from or susceptible to influenzainfection are tested for antibodies to inventive binding agents priorto, during, or after administration of inventive pharmaceuticalcompositions. In some embodiments, subjects having such antibodies arenot administered pharmaceutical compositions comprising inventivebinding agents. In some embodiments, an appropriate dose ofpharmaceutical composition and/or binding agent is selected based ondetection (or lack thereof) of such antibodies.

In some embodiments, selection of a particular subject for treatment,particular binding agent or composition for administration, and/orparticular dose or regimen for administration, is memorialized, forexample in a written, printed, or electronic storage form.

Inventive compositions may be administered prior to or after developmentof one or more symptoms of influenza infection.

The invention encompasses treatment and/or prevention (e.g.,vaccination) of influenza infections by administration of agents and/orcompositions described herein. In some embodiments, treatment ofinfluenza infections according to the present invention is accomplishedby administration of a vaccine. To date, although significantaccomplishments have been made in the development of influenza vaccines,there is room for further improvement. The present invention providesvaccines comprising inventive binding agents (e.g., HA polypeptides,particularly H2 HA polypeptides, LSBAs, UTBAs, UTBSAs, etc.), andparticularly comprising binding agents that bind to umbrella glycans(e.g., α2-6 linked umbrella glycans such as, for example, long α2-6sialylated glycans). In some embodiments, a composition is substantiallyfree of agents that preferentially bind to non-umbrella topologyglycans. In some such embodiments, pharmaceutical compositions containnot more than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of an agent that bindsto HA receptor glycans other than umbrella topology glycans.

To give but one example, the H2N2 influenza subtype stopped circulatingin humans by 1968, however H2 subtype viruses are occasionally isolatedfrom swine and avian species. The circulation of avian H2 strains indomestic birds and pigs increase the risk of human exposure to theseviruses and reintroduction of the viruses to the human population. Sucha reintroduction could lead to a significant global health threat giventhe lack of pre-existing immunity in a huge subset of the humanpopulation born after 1968. The present invention provides, among otherthings, vaccines comprising H2 HA polypeptides for treatment and/orprevention of influenza infections.

In some embodiments, the present invention provides for vaccines and theadministration of these vaccines to a human subject (e.g., to anindividual suffering from or susceptible to influenza infection). Insome embodiments, vaccines are compositions comprising one or more ofthe following: (1) inactivated virus, (2) live attenuated influenzavirus, for example, replication-defective virus, (3) inventive bindingagent (e.g., HA polypeptides and/or polypeptide variants, LSBAs, UTBAs,UTBSAs, etc.)), (4) nucleic acid encoding binding agent polypeptide(e.g., HA polypeptide) or characteristic or biologically active portionthereof, (5) DNA vector that encodes inventive binding agent polypeptide(e.g., HA polypeptide) or characteristic or biologically active portionthereof, and/or (6) expression system, for example, cells expressing oneor more influenza proteins to be used as antigens, and/or virus-likeparticles.

Thus, in some embodiments, the present invention provides inactivatedflu vaccines. In some embodiments, inactivated flu vaccines comprise oneof three types of antigen preparation: inactivated whole virus,sub-virions where purified virus particles are disrupted with detergentsor other reagents to solubilize the lipid envelope (“split” vaccine) orpurified HA polypeptide (“subunit” vaccine). In some embodiments, viruscan be inactivated by treatment with formaldehyde, beta-propiolactone,ether, ether with detergent (such as TWEEN-80), cetyl trimethyl ammoniumbromide (CTAB) and Triton N101, sodium deoxycholate and tri(n-butyl)phosphate. Inactivation can occur after or prior to clarification ofallantoic fluid (from virus produced in eggs); the virions are isolatedand purified by centrifugation (Nicholson et al., eds., Textbook ofInfluenza, Blackwell Science, Malden, Mass., 1998). To assess thepotency of the vaccine, the single radial immunodiffusion (SRD) test canbe used (Schild et al., Bull. World Health Organ., 52:43-50 & 223-31,1975; Mostow et al., J. Clin. Microbiol., 2:531, 1975; incorporatedherein by reference). In some embodiments, the present inventionprovides virus-like particles (VLPs) that are useful for vaccines. Ingeneral, VLPs comprise multiple copies of a protein antigen that, whenassembled together, mimic the conformation of a native virus. In someembodiments, VLPs contain repetitive high density displays of influenzavirus surface proteins (e.g., HA polypeptides in accordance with thepresent invention) which present conformational epitopes that can elicitstrong T cell and/or B cell immune responses. Since VLPs do not containany viral genetic material, they may be safer than attenuated viruses invaccine compositions. VLPs can be produced in a variety of cell culturesystems including mammalian cell lines, insect cell lines, yeast, plantcells, etc. For a general discussion of VLPs, see, e.g., published PCTapplications WO 02/000885, WO 05/020889, WO 06/108226, WO 07/130327, WO07/130330, WO 08/005777, WO 08/040060, WO 08/054535, WO 08/061243, WO08/094197, WO 08/094200, WO 08/148104, WO 09/009876, WO 09/012489, WO10/006452, and US patent application publication 2005/0009008, all ofwhich are incorporated herein by reference.

In some embodiments, a VLP in accordance with the invention is aspecialized VLP called a lipoparticle. In general, lipoparticles arestable, highly purified, homogeneous VLPs that are engineered to containhigh concentrations of a conformationally intact membrane protein ofinterest. In some embodiments, lipoparticles in accordance with thepresent invention contain influenza envelope proteins and/or otherinfluenza antigens.

The present invention also provides live, attenuated flu vaccines, andmethods for attenuation are well known in the art. In some embodiments,attenuation is achieved through the use of reverse genetics, such assite-directed mutagenesis.

In some embodiments, influenza virus for use in vaccines is grown ineggs, for example, in embryonated hen eggs, in which case the harvestedmaterial is allantoic fluid. Alternatively or additionally, influenzavirus may be derived from any method using tissue culture to grow thevirus. Suitable cell substrates for growing the virus include, forexample, dog kidney cells such as MDCK or cells from a clone of MDCK,MDCK-like cells, monkey kidney cells such as AGMK cells including Verocells, cultured epithelial cells as continuous cell lines, 293T cells,BK-21 cells, CV-1 cells, or any other mammalian cell type suitable forthe production of influenza virus (including upper airway epithelialcells) for vaccine purposes, readily available from commercial sources(e.g., ATCC, Rockville, Md.). Suitable cell substrates also includehuman cells such as MRC-5 cells. Suitable cell substrates are notlimited to cell lines; for example primary cells such as chicken embryofibroblasts are also included.

In some embodiments, vaccines further comprise one or more adjuvants.Any adjuvant may be used in accordance with the present invention. Alarge number of adjuvants are known; a useful compendium of many suchcompounds is prepared by the National Institutes of Health and can befound on the internet(www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf). See also Allison(1998, Dev. Biol. Stand., 92:3-11; incorporated herein by reference),Unkeless et al. (1998, Annu. Rev. Immunol., 6:251-281; incorporatedherein by reference), and Phillips et al. (1992, Vaccine, 10:151-158;incorporated herein by reference). Hundreds of different adjuvants areknown in the art and could be employed in the practice of the presentinvention. For example, aluminum salts (e.g., aluminum hydroxide,aluminum phosphate, etc., Baylor et al., Vaccine, 20:S18, 2002) andmonophosphoryl lipid A (MPL; Ribi et al., (1986, Immunology andImmunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY, p407, 1986) can be used as adjuvants in human vaccines. Alternatively oradditionally, exemplary adjuvants that can be utilized in accordancewith the invention include cytokines, calcium phosphate, microbialadjuvants (e.g., immunomodulatory DNA sequences that include CpG motifs;endotoxins such as monophosphoryl lipid A (Ribi et al., 1986, Immunologyand Immunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY,p 407, 1986; incorporated herein by reference); exotoxins such ascholera toxin, E. coli heat labile toxin, and pertussis toxin; muramyldipeptide, etc.); oil-emulsion and emulsifier-based adjuvants (e.g.,Freund's Adjuvant, SAF, etc.); particulate adjuvants (e.g., liposomes,biodegradable microspheres, etc.); synthetic adjuvants (e.g., nonionicblock copolymers, muramyl peptide analogues, polyphosphazene, syntheticpolynucleotides, etc.); polymers (e.g., polyphosphazenes; described inU.S. Pat. No. 5,500,161, which is incorporated herein by reference),Q57, squalene, and/or tetrachlorodecaoxide.

Alternatively or additionally, new compounds are currently being testedas adjuvants in human vaccines, such as MF59 (Chiron Corp.,http://www.chiron.com/investors/pressreleases/2005/051028.html), CPG7909 (Cooper et al., Vaccine, 22:3136, 2004; incorporated herein byreference), and saponins, such as QS21 (Ghochikyan et al., Vaccine,24:2275, 2006; incorporated herein by reference).

Additionally, some adjuvants are known in the art to enhance theimmunogenicity of influenza vaccines, such aspoly[di(carboxylatophenoxy)phosphazene] (PCCP; Payne et al., Vaccine,16:92, 1998; incorporated herein by reference), interferon-γ (Cao etal., Vaccine, 10:238, 1992; incorporated herein by reference), blockcopolymer P1205 (CRL1005; Katz et al., Vaccine, 18:2177, 2000;incorporated herein by reference), interleukin-2 (IL-2; Mbwuike et al.,Vaccine, 8:347, 1990; incorporated herein by reference), and polymethylmethacrylate (PMMA; Kreuter et al., J. Pharm. Sci., 70:367, 1981;incorporated herein by reference).

In some embodiments, pharmaceutical compositions do not includeadjuvants (e.g., provided compositions are essentially free ofadjuvants). In some embodiments, pharmaceutical compositions do notinclude an alum adjuvant (e.g., provided compositions are essentiallyfree of alum).

In addition to vaccines, the present invention provides othertherapeutic compositions useful in the treatment and/or vaccination ofviral infections. In some embodiments, treatment and/or vaccination isaccomplished by administration of an agent that interferes withexpression or activity of an HA polypeptide.

In some embodiments, the present invention provides pharmaceuticalcompositions comprising antibodies or other agents related to providedpolypeptides. For example, the invention provides compositionscontaining antibodies recognize virus particles containing a particularHA polypeptide (e.g., an HA polypeptide that binds to umbrella glycans),nucleic acids (such as nucleic acid sequences complementary to HAsequences, which can be used for RNAi), glycans that compete for bindingto HA receptors, small molecules or glycomometics that compete theglycan-HA polypeptide interaction, or any combination thereof. In someembodiments, collections of different agents, having diverse structuresare utilized. In some embodiments, therapeutic compositions comprise oneor more multivalent agents. In some embodiments, treatment comprisesurgent administration shortly after exposure or suspicion of exposure.

In general, a pharmaceutical composition will include a therapeuticagent in addition to one or more inactive agents such as a sterile,biocompatible carrier including, but not limited to, sterile water,saline, buffered saline, or dextrose solution. Alternatively oradditionally, a composition may comprise a pharmaceutically acceptableexcipient, which, as used herein, includes any and all solvents,dispersion media, diluents, or other liquid vehicles, dispersion orsuspension aids, disintegrating agents, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, bufferingagents, solid binders, granulating agents, lubricants, coloring agents,sweetening agents, flavoring agents, perfuming agents, and the like, assuited to the particular dosage form desired. Remington's The Scienceand Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro, (Lippincott,Williams & Wilkins, Baltimore, Md., 2006; incorporated herein byreference) discloses various excipients used in formulatingpharmaceutical compositions and known techniques for the preparationthereof. Except insofar as any conventional excipient medium isincompatible with a substance or its derivatives, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisinvention.

In some embodiments, the therapeutic agent present in an inventivepharmaceutical composition will consist of one or more binding agents asdescribed herein. In some embodiments, an inventive pharmaceuticalcomposition contains a binding agent (e.g., an HA polypeptide, LSBA,UTBA, UTSBA, etc.) that binds to umbrella topology glycans (and/or toumbrella topology glycan mimics). In some such embodiments, theinventive composition is substantially free of related agents (e.g., ofother HA polypeptides, etc.) that do not bind to umbrella-topologyglycans. In some such embodiments, the inventive pharmaceuticalcompositions contains not more than 50%, 40%, 30%, 20%, 10%, 5%, or 1%of an agent that binds to HA receptor glycans other than umbrellatopology glycans.

In some embodiments, a pharmaceutical composition will include atherapeutic agent that is encapsulated, trapped, or bound within a lipidvesicle, a bioavailable and/or biocompatible and/or biodegradablematrix, or other microparticle.

In some embodiments, a provided pharmaceutical composition will includea binding agent (e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.) thatis not aggregated. For example, in some embodiments, less than 1%, 2%,5%, 10%, 20%, or 30%, by dry weight or number, of the binding agent ispresent in an aggregate.

In some embodiments, a provided pharmaceutical composition will includea binding agent (e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.) thatis not denatured. For example, in some embodiments, less than 1%, 2%,5%, 10%, 20%, or 30%, by dry weight or number, of the UTSBA administeredis denatured.

In some embodiments, a provided pharmaceutical composition will includea binding agent (e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.) thatis not inactive. For example, in some embodiments, less than 1%, 2%, 5%,10%, 20%, or 30%, by dry weight or number, of the UTSBA administered isinactive.

In some embodiments, inventive pharmaceutical compositions areformulated to reduce immunogenicity of provided binding agents. Forexample, in some embodiments, a provided binding agent is associatedwith (e.g., bound to) an agent, such as polyethylene glycol and/orcarboxymethyl cellulose, that masks its immunogenicity. In someembodiments, a provided binding agent has additional glycosylation thatreduces immunogenicity.

Pharmaceutical compositions of the present invention may be administeredeither alone or in combination with one or more other therapeutic agentsincluding, but not limited to, vaccines and/or antibodies. By “incombination with,” it is not intended to imply that the agents must beadministered at the same time or formulated for delivery together,although these methods of delivery are within the scope of theinvention. In general, each agent will be administered at a dose and ona time schedule determined for that agent. Additionally, the inventionencompasses the delivery of the inventive pharmaceutical compositions incombination with agents that may improve their bioavailability, reduceor modify their metabolism, inhibit their excretion, or modify theirdistribution within the body. Although the pharmaceutical compositionsof the present invention can be used for treatment of any subject (e.g.,any animal) in need thereof, they are most preferably used in thetreatment of humans. In some embodiments, inventive pharmaceuticalcompositions and/or binding agents are administered in combination withone or more of an anti-viral agent (e.g., Oseltamivir [tamiflu],Zanamavir [Relenza], etc.) and/or a sialidase.

Pharmaceutical compositions may be administered using any amount and anyroute of administration effective for treatment and/or vaccination. Theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe infection, the particular composition, its mode of administration,its mode of activity, and the like. Pharmaceutical compositions aretypically formulated in dosage unit form for ease of administration anduniformity of dosage. It will be understood, however, that the totaldaily usage of the compositions of the present invention will be decidedby the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any particularsubject or organism will depend upon a variety of factors including thedisorder being treated and/or vaccinated and the severity of thedisorder; the activity of the specific vaccine composition employed; thehalf-life of the composition after administration; the age, body weight,general health, sex, and diet of the subject; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors, well known in the medical arts.

Pharmaceutical compositions of the present invention may be administeredby any route. In some embodiments, pharmaceutical compositions of thepresent invention are administered by a variety of routes, includingoral (PO), intravenous (IV), intramuscular (IM), intra-arterial,intramedullary, intrathecal, subcutaneous (SQ), intraventricular,transdermal, interdermal, intradermal, rectal (PR), vaginal,intraperitoneal (IP), intragastric (IG), topical (e.g., by powders,ointments, creams, gels, lotions, and/or drops), mucosal, intranasal,buccal, enteral, vitreal, sublingual; by intratracheal instillation,bronchial instillation, and/or inhalation; as an oral spray, nasalspray, and/or aerosol, and/or through a portal vein catheter.

In general, the most appropriate route of administration will dependupon a variety of factors including the nature of the agent beingadministered (e.g., its stability upon administration), the condition ofthe subject (e.g., whether the subject is able to tolerate a particularmode of administration), etc. In specific embodiments, pharmaceuticalcompositions may be administered intranasally. In specific embodiments,pharmaceutical compositions may be administered by intratrachealinstillation. In specific embodiments, pharmaceutical compositions maybe administered by bronchial instillation. In specific embodiments,pharmaceutical compositions may be administered by inhalation. Inspecific embodiments, pharmaceutical compositions may be administered asa nasal spray. In specific embodiments, pharmaceutical compositions maybe administered mucosally. In specific embodiments, pharmaceuticalcompositions may be administered orally. In specific embodiments,pharmaceutical compositions may be administered by intravenousinjection. In specific embodiments, pharmaceutical compositions may beadministered by intramuscular injection. In specific embodiments,pharmaceutical compositions may be administered by subcutaneousinjection. At present the oral or nasal spray or aerosol route (e.g., byinhalation) is most commonly used to deliver therapeutic agents directlyto the lungs and respiratory system. However, the invention encompassesthe delivery of the inventive composition by any appropriate routetaking into consideration likely advances in the sciences of drugdelivery.

In some embodiments, preparations for inhaled or aerosol deliverycomprise a plurality of particles. In some embodiments, suchpreparations have a mean particle size of about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, about 12, or about 13microns. In some embodiments, preparations for inhaled or aerosoldelivery are formulated as a dry powder. In some embodiments,preparations for inhaled or aerosol delivery are formulated as a wetpowder, for example through inclusion of a wetting agent. In someembodiments, the wetting agent is selected from the group consisting ofwater, saline, or other liquid of physiological pH.

In some embodiments, inventive compositions are administered as drops tothe nasal or buccal cavity. In some embodiments, a dose may comprise aplurality of drops (e.g., 1-100, 1-50, 1-20, 1-10, 1-5, etc.)

In some embodiments, inventive compositions are administered using adevice that delivers a metered dosage of composition (e.g., of bindingagent).

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. No. 4,886,499, U.S. Pat. No. 5,190,521, U.S. Pat.No. 5,328,483, U.S. Pat. No. 5,527,288, U.S. Pat. No. 4,270,537, U.S.Pat. No. 5,015,235, U.S. Pat. No. 5,141,496, U.S. Pat. No. 5,417,662;all of which are incorporated herein by reference. Intradermalcompositions may also be administered by devices which limit theeffective penetration length of a needle into the skin, such as thosedescribed in WO99/34850, incorporated herein by reference, andfunctional equivalents thereof. Also suitable are jet injection deviceswhich deliver liquid vaccines to the dermis via a liquid jet injector orvia a needle which pierces the stratum corneum and produces a jet whichreaches the dermis. Jet injection devices are described for example inU.S. Pat. No. 5,480,381, U.S. Pat. No. 5,599,302, U.S. Pat. No.5,334,144, U.S. Pat. No. 5,993,412, U.S. Pat. No. 5,649,912, U.S. Pat.No. 5,569,189, U.S. Pat. No. 5,704,911, U.S. Pat. No. 5,383,851, U.S.Pat. No. 5,893,397, U.S. Pat. No. 5,466,220, U.S. Pat. No. 5,339,163,U.S. Pat. No. 5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No.5,064,413, U.S. Pat. No. 5,520,639, U.S. Pat. No. 4,596,556, U.S. Pat.No. 4,790,824, U.S. Pat. No. 4,941,880, U.S. Pat. No. 4,940,460, WO97/37705 and WO 97/13537; all of which are incorporated herein byreference. Also suitable are ballistic powder/particle delivery deviceswhich use compressed gas to accelerate vaccine in powder form throughthe outer layers of the skin to the dermis. Additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

General considerations in the formulation and manufacture ofpharmaceutical agents may be found, for example, in Remington'sPharmaceutical Sciences, 19^(th) ed., Mack Publishing Co., Easton, Pa.,1995.

Inventive pharmaceutical compositions may be administered in any doseappropriate to achieve a desired outcome. In some embodiments, thedesired outcome is reduction in intensity, severity, and/or frequency,and/or delay of onset of one or more symptoms of influenza infection.

In some embodiments, inventive pharmaceutical compositions areformulated to administer a dose of binding agent effective to competewith influenza HA for binding to umbrella topology glycans. In someembodiments, such binding by influenza HA is reduced afteradministration of one or more doses of an inventive composition ascompared with its level absent such administration. In some embodiments,inventive pharmaceutical compositions are formulated to administer adose of binding agent effective to saturate at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or more than 99% ormore HA binding sites (e.g., HA binding sites containing umbrellatopology glycans) present in the subject (e.g., in the respiratory tractof the subject) receiving the composition.

In some embodiments, pharmaceutical compositions may be administered atdosage levels sufficient to deliver from about 0.001 mg/kg to about 100mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg of a therapeutic agent per subject bodyweight per day to obtain a desired therapeutic effect. A desired dosagemay be delivered to a subject only once. A desired dosage may bedelivered more than three times per day, three times per day, two timesper day, once per day, every other day, every third day, every week,every two weeks, every three weeks, every four weeks, every two months,every six months, every twelve months, every two years, every threeyears, every four years, every five years, every 10 years, or every 20years. In some embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or moreadministrations).

It will be appreciated that compositions in accordance with the presentinvention can be employed in combination therapies. The particularcombination of therapies (e.g., therapeutics or procedures) to employ ina combination regimen will take into account compatibility of thedesired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will be appreciated that the therapiesemployed may achieve a desired effect for the same purpose (for example,an agent useful for treating, preventing, and/or delaying the onset ofinfluenza infection may be administered concurrently with another agentuseful for treating, preventing, and/or delaying the onset of influenzainfection), or they may achieve different effects (e.g., control of anyadverse effects). The invention encompasses delivery of pharmaceuticalcompositions in combination with agents that may improve theirbioavailability, reduce and/or modify their metabolism, inhibit theirexcretion, and/or modify their distribution within the body.

Pharmaceutical compositions in accordance with the present invention maybe administered either alone or in combination with one or more othertherapeutic agents. By “in combination with,” it is not intended toimply that the agents must be administered at the same time and/orformulated for delivery together, although these methods of delivery arewithin the scope of the invention. Compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. In will be appreciated thattherapeutically active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, each agent will be administeredat a dose and/or on a time schedule determined for that agent.

In general, it is expected that agents utilized in combination with beutilized at levels that do not exceed the levels at which they areutilized individually. In some embodiments, the levels utilized incombination will be lower than those utilized individually.

In some embodiments, pharmaceutical compositions are administered incombination with one or more of an anti-viral agent (e.g., Oseltamivir[tamiflu], Zanamavir [Relenza], etc.) and/or a sialidase.

Diagnostics/Kits

The present invention provides kits for detecting binding agents (e.g.,HA polypeptides, LSBAs, UTBAs, UTSBAs, etc), and particular fordetecting binding agents with particular glycan binding characteristics(e.g., binding to umbrella glycans, to α2-6 sialylated glycans, to longα2-6 sialylated glycans, etc.) in pathological samples, including, butnot limited to, blood, serum/plasma, peripheral blood mononuclearcells/peripheral blood lymphocytes (PBMC/PBL), sputum, urine, feces,throat swabs, dermal lesion swabs, cerebrospinal fluids, cervicalsmears, pus samples, food matrices, and tissues from various parts ofthe body such as brain, spleen, and liver. The present invention alsoprovides kits for detecting binding agents (e.g., HA polypeptides,LSBAs, UTBAs, UTSBAs, etc) of interest in environmental samples,including, but not limited to, soil, water, and flora. Other samplesthat have not been listed may also be applicable.

In some embodiments, the present invention provides kits for detectingHA polypeptides as described herein whether or not such polypeptides arebinding agents.

In some embodiments, inventive kits may include one or more agents thatspecifically detect binding agents (e.g., HA polypeptides, LSBAs, UTBAs,UTSBAs, etc) with particular glycan binding characteristics. Suchdetecting agents may include, for example, antibodies that specificallyrecognize certain binding agents (e.g., binding agents that bind toumbrella glycans and/or to α2-6 sialylated glycans and/or to long α2-6sialylated glycans), which can be used to specifically detect suchbinding agents by ELISA, immunofluorescence, and/or immunoblotting.

Antibodies that bind to HA polypeptides (e.g., to provided HApolypeptides such as HA polypeptide variants) can also be used in virusneutralization tests, in which a sample is treated with antibodyspecific to HA polypeptides of interest, and tested for its ability toinfect cultured cells relative to untreated sample. If the virus in thatsample contains such HA polypeptides, the antibody will neutralize thevirus and prevent it from infecting the cultured cells. Alternatively oradditionally, such antibodies can also be used in HA-inhibition tests,in which the HA protein is isolated from a given sample, treated withantibody specific to a particular HA polypeptide or set of HApolypeptides, and tested for its ability to agglutinate erythrocytesrelative to untreated sample. If the virus in the sample contains suchan HA polypeptide, the antibody will neutralize the activity of the HApolypeptide and prevent it from agglutinating erythrocytes (Harlow &Lane, Antibodies: A Laboratory Manual, CSHL Press, 1988;www.who.int/csr/resources/publications/influenza/WHO_CDS_CSR_NCS2002_(—)5/en/index.html;www.who.int/csr/disease/avian_influenza/guidelines/labtests/en/index.html).In other embodiments, such agents may include nucleic acids thatspecifically bind to nucleotides that encode particular HA polypeptidesand that can be used to specifically detect such HA polypeptides byRT-PCR or in situ hybridization(www.who.int/csr/resources/publications/influenza/WHO_CDS_CSR_NCS2002_(—)5/en/index.html;www.who.int/csr/disease/avian_influenza/guidelines/labtests/en/index.html).In some embodiments, nucleic acids which have been isolated from asample are amplified prior to detection. In some embodiments, diagnosticreagents can be detectably labeled.

The present invention also provides kits containing reagents accordingto the invention for the generation of influenza viruses and vaccines.Contents of the kits include, but are not limited to, expressionplasmids containing HA nucleotides (or characteristic or biologicallyactive portions) encoding HA polypeptides of interest (or characteristicor biologically active portions). Alternatively or additionally, kitsmay contain expression plasmids that express HA polypeptides of interest(or characteristic or biologically active portions). Expression plasmidscontaining no virus genes may also be included so that users are capableof incorporating HA nucleotides from any influenza virus of interest.Mammalian cell lines may also be included with the kits, including butnot limited to, Vero and MDCK cell lines. In some embodiments,diagnostic reagents can be detectably labeled.

In some embodiments, kits for use in accordance with the presentinvention may include, a reference sample, instructions for processingsamples, performing the test, instructions for interpreting the results,buffers and/or other reagents necessary for performing the test. In someembodiments the kit can comprise a panel of antibodies.

In some embodiments of the present invention, glycan arrays, asdiscussed above, may be utilized as diagnostics and/or kits.

In some embodiments, inventive glycan arrays and/or kits are used toperform dose response studies to assess binding of HA polypeptides toumbrella glycans at multiple doses (e.g., as described herein). Suchstudies give particularly valuable insight into the bindingcharacteristics of tested HA polypeptides, and are particularly usefulto assess specific binding. Dose response binding studies of this typefind many useful applications. To give but one example, they can behelpful in tracking the evolution of binding characteristics in arelated series of HA polypeptide variants, whether the series isgenerated through natural evolution, intentional engineering, or acombination of the two.

In some embodiments, inventive glycan arrays and/or kits are used toinduce, identify, and/or select binding agents (e.g., HA polypeptides,and/or HA polypeptides such as HA polypeptide variants) having desiredbinding characteristics. For instance, in some embodiments, inventiveglycan arrays and/or kits are used to exert evolutionary (e.g.,screening and/or selection) pressure on a population of polypeptidebinding agents (e.g., HA polypeptides).

The present invention provides kits for administration of inventivepharmaceutical compositions. For example, in some embodiments, theinvention provides a kit comprising at least one dose of a bindingagent. In some embodiments, the invention provides a kit comprising aninitial unit dose and a subsequent unit dose of a binding agent. In somesuch embodiments, the initial unit dose is greater than the subsequentunit dose or wherein the two doses are equal.

In some embodiments, inventive kits (particularly those foradministration of inventive pharmaceutical compositions) comprise atleast one component of a delivery device, e.g., an inhaler. In some suchembodiments, the invention provides a kit comprising at least onecomponent of a delivery device, e.g., an inhaler and a dose of an of abinding agent.

In some embodiments, provided kits comprise instructions for use.

EXEMPLIFICATION Example 1 H2N2 HA Variants

The 20th century witnessed three influenza pandemics: the Spanish flu of1918 (H1N1), the Asian flu of 1957-58 (H2N2) and the Hong Kong flu of1967-68 (H3N2). Among these subtypes the H1N1 and H3N2 continue tocirculate in the human population leading to epidemic outbreaks annuallyand the H1N1 subtype was responsible for the 2009 ‘swine flu’ pandemic(2009 H1N1). The H2N2 subtype had stopped circulating in humans by 1968,however H2 subtype viruses are occasionally isolated from swine andavian species. The circulation of avian H2 strains in domestic birds andpigs increase the risk of human exposure to these viruses andreintroduction of the viruses to the human population. Such areintroduction may pose a significant global health threat given thelack of pre-existing immunity in a huge subset of the human populationborn after 1968.

One of the main steps in the evolution of a pandemic influenza virus isthe acquisition of genetic changes that enable it to adapt to the humanhost in order to replicate efficiently and transmit rapidly resulting inwidespread and sustained disease in humans. An important first step inthe host infection by the virus is the binding of the viral surfaceglycoprotein hemagglutinin (HA) to sialylated glycan receptors, complexglycans terminated by N-acetylneuraminic acid (Neu5Ac) expressed on thehost cell surface. Glycans terminating in Neu5Ac that is α2→6-linked tothe penultimate sugar are predominantly expressed in human upperrespiratory epithelia and serve as receptors for human-adapted influenzaA viruses (henceforth referred to as human receptors). On the otherhand, glycans terminating in Neu5Ac that is a2→3-linked to thepenultimate sugar residue, serve as receptors for the avian-adaptedinfluenza viruses (henceforth referred to as avian receptors).

The molecular interactions of HA with avian and human receptors havebeen captured using a topology-based definition of glycan receptors.Glycan array platforms comprised of representative avian and humanreceptors have been widely employed to study the glycan receptor bindingof HAs and whole viruses. The relative binding affinities ofrecombinantly expressed HAs from avian—(such as H1N1 and H5N1) andhuman-adapted (such as H1N1 and H3N2) viruses to avian and humanreceptors have been quantified by analyzing these HAs (or whole viruses)in a dose-dependent manner on glycan array platforms. Furthermore, theglycan array binding properties of the HAs have been shown to correlatewith their binding to physiological glycan-receptors in humanrespiratory tissues. It has been shown that the human receptor-bindingaffinity of H1N1 HAs correlated with the efficiency of airborne viraltransmission in the ferret animal model, which is an established modelto evaluate viral transmissibility in humans. Such a relationship hasyet to be shown for the H2N2 subtype.

Previous structural and biochemical studies have provided insights intointeractions of the receptor binding site (RBS) of HA with avian andhuman receptors for both wild type (WT) and mutant forms of HA derivedfrom the 1957-58 H2N2 pandemic strains. However, it has been recentlydemonstrated that changes in the interactions between amino acids withinand proximal to the RBS, arising from substitutions due to antigenicdrift or reassortment, have profound effects on HA-glycan interactionswhich in turn influences the glycan binding affinity of HA. Thisobservation is particularly relevant to HA from recent avian-H2 strainsthat have diverged considerably in sequence compared to the HA sequenceof the pandemic H2N2 strains. Therefore in order to monitor changes inthe recent avian H2-subtype viruses that would possibly lead to theirhuman-adaptation, it is important to understand the mutations in theirHA that would confer human receptor-binding affinity that isquantitatively in the same range as that of HA from the 1957-58human-adapted H2N2 pandemic viruses.

In the present example, we systematically analyzed the effects ofmutations in the glycan RBS of pandemic and recent avian H2N2 HAs ontheir respective glycan-binding specificities. The HA from arepresentative 1957-58 pandemic H2N2 strain, A/Albany/6/58 (Alb58), waschosen as a reference human-adapted HA. The HA from a representativeavian H2N2 virus, A/Chicken/Pennsylvania/2004 (CkPA04), which is amongthe most recent strains isolated from birds was also evaluated in thisstudy. We first characterized the glycan receptor-binding affinity andhuman respiratory tissue binding properties of these avian- andhuman-adapted H2N2 HAs. The glycan receptor-binding affinity of HA isquantitatively defined using an apparent binding constant K_(d)′ thattakes into account the cooperativity and avidity in the multivalentHA-glycan interactions as described previously. Next, usinghomology-based structural models of Alb58 HA-human receptor and CkPA04HA-avian receptor complexes we analyzed the RBS of these HAs anddesigned and evaluated mutations in CkPA04 HA that would make its humanreceptor binding affinity in the same range as that of Alb58 HA.

Characterization of Glycan Receptor-Binding Specificity of Alb58 HA.

We have previously developed a dose-dependent glycan array binding assayto quantitatively characterize glycan receptor binding affinity of HA bycalculating an apparent binding constant Kd′. Alb58 HA was recombinantlyexpressed and analyzed using this assay. Alb58 HA bound with highaffinity to the representative human receptors, 6′SLN (Kd′˜35 pM) and6′SLN-LN (Kd′˜5 pM) (FIG. 5A). Notably, the binding affinity of Alb58 HAto 6′SLN-LN is in the same range as that of the pandemic H1N1 (A/SouthCarolina/1/1918 or SC18) HA. However unlike SC18 HA, surprisingly, Alb58HA also showed substantial binding to the representative avian receptors3′SLN-LN (Kd′˜1.5 nM) and 3′SLN-LN-LN (Kd′˜1 nM) on the glycan array(FIG. 5A). Staining of Alb58 HA on human upper respiratory trachealtissue sections revealed extensive binding of the protein to the apicalside (FIG. 5B) and thus correlated with its high affinity binding tohuman receptors. Additionally, the substantial a2→3 sialylated glycanbinding of Alb58 observed in the glycan array assay was also reflectedin its binding to the human deep lung alveolar tissue (FIG. 5B) thatpredominantly expresses these glycans.

Previous studies have pointed to the roles played by the amino acids inpositions 226 and 228 in the RBS of H2N2 HAs in governing the glycanreceptor binding specificity. The observation includes the fact that HAfrom most human H2N2 isolates has Leu226 and Ser228 within its RBS,whereas HA from most avian H2 isolates has Gln226 and Gly228. Tounderstand the roles of these residues on the quantitative glycanreceptor binding affinity of Alb58 HA, three mutant forms of Alb58 weredesigned. Two of these mutants possessed a single amino acid change,Leu226→Gln (Alb58-QS mutant) and Ser228→Gly (Alb58-LG). The third mutantcarried two amino acid changes, Leu226→Gln/Ser228→Gly (Alb58-QG).

Alb58-LG mutant retained the human receptor binding specificity of theWT Alb58 HA but showed a complete loss in the avian receptor binding inthe dose-dependent direct binding assay (FIG. 6A). On the other hand,Alb58-QG mutant showed a complete loss in human receptor binding and butdisplayed a substantial binding to avian receptors in contrast to Alb58HA (FIG. 6B). Surprisingly, Alb58-QS mutant exhibited little to nobinding to either the avian or human glycan receptor (FIG. 6C). Circulardichroism analysis of Alb58-QS ruled out the possibility of Alb58-QSbeing misfolded. A homology-based structural model of the Alb58-QSmutant was constructed to investigate the molecular basis of theobserved biochemical binding property. Analysis of the glycanreceptor-binding site of this mutant in the model showed that Ser228 ispositioned to form a hydrogen bond with Gln226 (FIG. 6D). Theinteraction between Gln226 and Ser228 potentially disrupts the favorablepositioning of Gln226 for optimal contact with avian receptor. Thisobservation offers an explanation for the loss of avian receptor bindingin the Alb58-QS mutant. Furthermore, the absence of contacts betweenGln226 and human receptor could explain the loss of human receptorbinding.

Mutations in RBS of CkPA04 and their Effects on its Glycan ReceptorBinding Specificity.

The dose-dependent glycan array binding of CkPA04 HA showed highaffinity binding to the representative avian receptors 3′SLN, 3′SLN-LNand 3′SLN-LN-LN with minimal binding to human receptors (FIG. 7A).Furthermore, the glycan array binding property of CkPA04 correlated withits extensive binding to the human alveolar tissues and minimal bindingto the apical side of the tracheal tissues (FIG. 7B).

To understand the molecular aspects of the H2 HA-glycan receptorinteraction, we constructed homology-based structural models of theCkPA04-avian (FIG. 8A) and the Alb58-human receptor complexes (FIG. 8B).Based on these structural models of CkPA04 and Alb58 HAs, the aminoacids positioned to interact with the glycan receptors were compared(Table 7).

TABLE 7 Comparison of key amino acids in the RBS of CkPA04 and Alb58 HAs136 137 153 155 156 183 186 187 189 190 193 194 222 226 228 CkPA04 S Q WT K H N D T E A L K Q G Alb58 S R W T K H N D T E T L K L S

In addition to the differences in 226 and 228 positions, there weredifferences in other positions including 137 and 193. The amino acids atpositions 137 and 193 are oriented to interact with Neu5Acα2→6Gal motifas well as sugars beyond this motif in the context of the human receptor(and potentially play a role in antigenic variations among currentstrains of H2 viruses; see discussion). These differences potentiallyimpinge on the human receptor binding of H2N2 HA. Notably, CkPA04 HAdiffers from earlier avian-adapted H2N2 HAs in the 137 and 193positions. Therefore, while the Gln226→Leu and Gly228→Ser substitutionswould make the RBS of earlier avian-adapted H2N2 HAs almost identical tothat of the pandemic Alb58 HA, additional amino acid changes arerequired in the more recent avian-adapted HAs, including CkPA04.

Based on the above analysis, three sets of mutations were progressivelymade on CkPA04 to improve its contacts with the human receptor. Thefirst mutant comprised of the two amino acid changeGln226→Leu/Gly228→Ser (CkPA04-LS). The second mutant, CkPA04-TLS,included an additional Ala193→Thr amino acid change in the CkPA04-LS HA.The third mutant, CkPA04-RTLS, was generated by introducing anadditional Gln137→Arg mutation in the CkPA04-TLS HA. These HA mutantswere recombinantly expressed and characterized in terms of theirquantitative glycan receptor binding affinity and human tissue bindingproperties.

CkPA04-LS showed decreased binding to avian receptors and substantialbinding to human receptors in comparison with CkPA04 (FIG. 9A).CkPA04-TLS showed substantially higher binding signals to both human andavian receptors when compared to CkPA04-LS (FIG. 9C). CkPA04-RTLS on theother hand showed increased binding signals to human receptor andsimilar binding signals to avian receptor as compared to CkPA04-LS (FIG.9E). The human respiratory tissue binding of these mutant H2 HAs was inagreement with their observed glycan array binding (FIG. 9B, 9D, 9F).The dose-dependant glycan binding data of the described HAs were used tocalculate Kd′ and n values (n˜1.3 for all the HAs) by fitting thebinding data to the Hill equation (for multivalent binding) and this wasthen used to generate theoretical binding curves to clearly distinguishthe relative binding affinities of WT and mutant H2 HAs torepresentative avian and human receptors (FIG. 10). The human receptorbinding affinity of CkPA04-LS (Kd′˜50 pM) was 10-fold lower than that ofthe Alb58 HA (Kd′˜5 pM). On the other hand the human receptor bindingaffinity of both CkPA04-TLS (Kd′˜3 pM) and CkPA04-RTLS (Kd′˜8 pM) wereseveral fold higher than that of CkPA04-LS and in the same range as thatof Alb58 HA. The avian receptor binding affinity of CkPA04-TLS (Kd′˜50pM) was in the same range as that of the WT CkPA04 HA (Kd′˜20 pM) andseveral fold higher than that of CkPA04-LS (Kd′˜220 pM) and CkPA04-RTLS(Kd′˜220 pM). Therefore, among the different mutants, CkPA04-RTLS wasthe closest to Alb58 HA in terms of its relative human to avian receptorbinding affinity. Based on our structural understanding, thisobservation is consistent with the fact that the RBS of CkPA04-RTLS andAlb58 were very similar to each other, including extended range contactswith the glycan receptor beyond the Neu5Ac linkage.

Our study highlights the value of integrating a systematic sequence andstructure analysis of HA-glycan molecular interactions and aquantitative binding assay to study the effects of these interactions onthe biochemical glycan receptor binding affinity of HA.

Previous studies have focused on amino acid substitutions in 226 and 228positions in the RBS of pandemic H2N2 HAs. Recently the glycanreceptor-binding properties of the Alb58 virus and the WT and mutantforms (with substitutions in 226 and 228 positions in HA) of a relatedpandemic H2N2 virus—A/El Salvador/2/57 (or ElSalv57) were characterizedby analyzing these whole viruses in a dose dependent fashion on theglycan array platform. The glycan receptor-binding properties of therecombinant Alb58 HA reported in the present study are in good agreementwith those obtained using the whole viruses. Our results further augmentthese observations by characterizing the effect of substitutions in the226 and 228 position on the quantitative glycan receptor bindingaffinity of Alb58 HA.

In addition to the previously noted 226 and 228 positions, oursystematic sequence and structural analysis of H2 HA-glycan complexesrevealed differences between CkPA04 and Alb58 HAs in other positions,such as 137 and 193. By progressively designing mutations in CkPA04 wehave demonstrated that substitutions at the 137 and 193 positions (inaddition to those in 226 and 228 positions) considerably alter theglycan receptor binding affinity. In fact, introducing these additionalamino acid changes (CkPA04-TLS and CkPA04-RTLS mutants) leads to a10-fold increase in the human receptor binding affinity compared to thatof the CkPA04-LS mutant and makes the affinity in the range of thatobserved for the pandemic H2N2 HA (Alb58). Therefore, monitoring themutations in these additional positions in the RBS is valuable forunderstanding changes in glycan receptor binding affinity of the H2 HAs.Moreover, these additional positions are also a part of antigenic loopsand hence are likely to undergo constant substitutions as a result ofantigenic drift in the H2 viruses to escape antibody neutralization.Monitoring these mutations also have important implications in vaccinedevelopment should a scenario arise wherein recent avian or swine H2viruses are able to gain a foothold in the human population.

The apparent binding constant Kd′ calculated in our study is usedprimarily to compare the relative binding affinities of differentrecombinant HAs by taking into account a defined spatial arrangement ofHA (that is fixed for all the HAs) relative to the glycans. Among thevarious factors that influence the efficient viral transmissibility inhumans we have shown in both the 1918 pandemic H1N1 and the recentlydeclared 2009 pandemic H1N1 that the binding affinity to the humanreceptors (quantified using Kd′) correlates with the transmissibility ofthe virus via respiratory droplets in ferrets. The human receptorbinding affinity of Alb58 HA being in the same range as that of the SC18HA taken together with the efficient respiratory droplet transmission ofthe Alb58 virus extends this correlation to the H2N2 viruses.Furthermore, given that Alb58 virus transmits efficiently viarespiratory droplets in ferrets, our results underscores the fact that acomplete switch from avian to human receptor binding is not the criticaldeterminant for human adaptation of influenza A virus HAs. Both thequantitative glycan array binding and human tissue binding results ofAlb58 HA show substantial avian receptor binding. Instead, it appearsthat the high affinity binding to human receptors is a common factorshared by H2 HA with that of other human-adapted virus subtypes (H1 andH3) and therefore this property appears to be an important determinantfor efficient human adaptation and transmission. In summary our studiesoffer valuable strategies to monitor the evolution of human-adaptivemutations in the HA of currently circulating avian H2 influenza Aviruses.

The present disclosure reports the first description of an H2 HApolypeptide characterized by the absolute and/or relative bidingaffinities reported herein. Now that the present disclosure hasestablished that it is possible to provide such H2 HA polypeptides,those of ordinary skill in the art will appreciate that other H2 HApolypeptides, e.g., containing one or more sequence variations ascompared with the specific sequences of H2 HA polypeptides explicitlytested herein, can be prepared that will similarly be characterized bysuch absolute and/or relative binding affinities. The present inventiontherefore provides H2 HA polypeptides characterized in that they showbinding to umbrella topology glycans with high affinity.

For example, in some embodiments, H2 HA polypeptide binding to umbrellaglycans is within a range of 10-fold or less (e.g., 9-fold, 8-fold,7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1.5-fold, etc.) of theaffinity for a wild type HA that mediates infection of a humans.

In some embodiments, H2 HA polypeptide binding to umbrella glycans hasan affinity of at least 25%, at least 30%, at least 35%, at least 40%,at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or atleast 100% of that observed under comparable conditions for a wild typeHA that mediates infection of humans (e.g., is human transmissible).

In some embodiments, H2 HA polypeptides show a signal for binding toumbrella topology glycans above about 400000 or more (e.g., above about500000, about 600000, about 700000, about 800000, etc) in a multivalentglycan array binding assay.

In some embodiments, H2 HA polypeptides show an affinity (Kd′) forumbrella-topology glycans within the range of about 1.5 nM to about 2pM. In some embodiments, H2 HA polypeptides show a Kd′ for binding tocone-topology glycans of about 100 pM or more (e.g., above about 200 pM,about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM,about 800 pM, about 900 pM, about 1 nM, about 1.1 nM, about 1.2 nM,about 1.3 nM, about 1.4 nM, about 1.5 nM, etc.) in binding assays. Insome embodiments, H2 HA polypeptides show a Kd′ of about 500 pM or less(e.g., below about 400 pM, about 300 pM, about 200 pM, about 100 pM,about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about40 pM, about 30 pM, about 20 pM, about 10 pM, about 5 pM, about 4 pM,about 3 pM, about 2 pM, etc.) for umbrella topology glycans and a Kd′ ofabout 100 pM or more (e.g., above about 200 pM, about 300 pM, about 400pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900pM, about 1 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM,about 1.5 nM, etc.) for cone topology glycans in binding assays.

In some embodiments, H2 HA polypeptides show a relative affinity forumbrella glycans vs cone glycans that is about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about20, about 30, about 40, about 50, about 60, about 70, about 80, about90, about 100, about 200, about 300, about 400, about 500, about 600,about 700, about 800, about 900, about 1000, about 2000, about 3000,about 4000, about 5000, about 6000, about 7000, about 8000, about 9000,about 10,000, up to about 100,000 or more. In some embodiments, H2 HApolypeptides show an affinity for umbrella topology glycans that isabout 100%, about 200%, about 300%, about 400%, about 500%, about 600%,about 700%, about 800%, about 900%, about 1000%, about 2000%, about3000%, about 4000%, about 5000%, about 6000%, about 7000%, about 8000%,about 9000%, about 10,000% or more than their affinity for cone topologyglycans.

In some embodiments, H2 HA polypeptides bind to at least about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95% or more of the glycans foundon HA receptors in human upper respiratory tract tissues (e.g.,epithelial cells).

In some embodiments, H2 HA polypeptides have an amino acid at aparticular residue (e.g., 137, 145, 186, 187, 189, 190, 192, 193, 222,225, 226, 228) that is predominantly present in the correspondinghuman-adapted HA (e.g., human-adapted H2 HA, such as those shown in FIG.1). In some embodiments, provided HA polypeptides such as HA polypeptidevariants (e.g., H2 HA polypeptides such as H2 HA polypeptide variants)have at least one amino acid substitution that is found in thecorresponding human-adapted HA (e.g., human-adapted H2 HA). In someembodiments, H2 HA polypeptides have a sequence that differs from thewild-type H2 HA sequence.

Materials and Methods

Homology Based Modeling of CkPA04 HA- and Alb58 HA-Glycan StructuralComplexes

The co-crystal structures of A/Singapore/1/57 H2N2 HA-human receptor(PDB ID: 2WR7) and A/ck/NewYork/91-avian receptor (PDB ID: 2WR2) wereused as templates to model the structural complexes of Alb58-humanreceptor and CkPA04-avian receptor respectively. Homology modeling wasperformed using the SWISS-MODEL web-based program (URL:http://swissmodel.expasy.org/SWISS-MODEL.html).

Cloning, Mutagenesis and Expression of HA

The Alb58 and CkPA04 plasmids were gifts from Dr. Terrence Tumpey andDr. Adolfo Garcia-Sastre respectively. The human and avian WT H2N2 HAgenes were subcloned into a pAcGP67A vector to generate pAcGp67-Alb58-HAand pAcGp67-CkPA04-HA respectively for baculovirus expression in insectcells. Using pAcGp67-CkPA04-HA as a template the gene was mutated toyield pAcGp67-LS-HA [Gln226Leu, Gly228Ser], pAcGp67-TLS-HA [Ala193Thr,Gln226Leu, Gly228Ser] and pAcGp67-RTLS-HA [Gln137Arg, Ala193Thr,Gln226Leu, Gly228Ser]. The primers for mutagenesis were designed usingPrimerX (http://bioinformatics.org/primerx/) and synthesized by IDT DNAtechnologies (Coralville, Iowa). The mutagenesis reaction was carriedout using the QuikChange Multi Site-Directed Mutagenesis Kit(Stratagene, C A) Alb58, CkPA04, CkPA04-LS, CkPA04-TLS and CkPA04-RTLSbaculoviruses were created from their respective plasmids, usingBaculogold system (BD Biosciences, CA) as per the manufacturer'sinstructions. The baculoviruses were used to infect 300 ml suspensioncultures of Sf9 cells (Invitrogen, Carlsbad, Calif.) cultured in Sf-900II SFM medium (Invitrogen, Carlsbad, Calif.). The infected cultures weremonitored and harvested 4-5 days post-infection. The soluble trimericform of HA was purified from the supernatant of infected cells usingmodification of the protocol described previously. In brief, thesupernatant was concentrated using Centricon Plus-70 centrifugal filters(Millipore, Billerica, Mass.) and the trimeric HA was recovered from theconcentrated cell supernatant using affinity chromatography with columnspacked with Ni-NTA beads (Qiagen, Valencia, Calif.). The fractionscontaining HA were pooled together and subjected to ultrafiltrationusing Amicon Ultra 100 K NMWL membrane filters (Millipore, Billerica,Mass.). The protein was reconstituted in PBS and concentrated. Thepurified protein concentration was determined using Bio-Rad's proteinassay (Bio-Rad, CA).

Dose Dependent Direct Glycan Array-Binding Assay

To investigate the multivalent HA-glycan interactions a streptavidinplate array comprising representative biotinylated a2→3 and a2→6sialylated glycans as described previously. The glycans 3′SLN, 3′SLN-LN,3′SLN-LN-LN are representative avian receptors. 6′SLN and 6′SLN-LN arerepresentative human receptors. LN corresponds to lactosamine(Galβ1-4GlcNAc) and 3′SLN and 6′SLN respectively correspond toNeu5Acα2-3 and Neu5Acα2-6 linked to LN. The biotinylated glycans wereobtained from the Consortium of Functional Glycomics through theirresource request program. Streptavidin-coated High Binding Capacity384-well plates (Pierce) were loaded to the full capacity of each wellby incubating the well with 50 μl of 2.4 μM of biotinylated glycansovernight at 4° C. Excess glycans were removed through extensive washingwith PBS.

The trimeric HA unit comprises of three HA monomers (and hence threeRBS, one for each monomer). The spatial arrangement of the biotinylatedglycans in the wells of the streptavidin plate array favors binding toonly one of the three HA monomers in the trimeric HA unit. Therefore inorder to specifically enhance the multivalency in the HA-glycaninteractions, the recombinant HA proteins were pre-complexed with theprimary and secondary antibodies in the ratio of 4:2:1(HA:primary:secondary). The identical arrangement of 4 trimeric HA unitsin the precomplex for all the HAs permits comparison between theirglycan binding affinities.

A stock solution containing appropriate amounts of Histidine tagged HAprotein, primary antibody (Mouse anti 6×His tag IgG) and secondaryantibody (HRP conjugated goat anti Mouse IgG (Santacruz Biotechnology)in the ratio 4:2:1 and incubated on ice for 20 min. Appropriate amountsof precomplexed stock HA were diluted to 250 μl with 1% BSA in PBS. 50μl of this precomplexed HA was added to each of the glycan-coated wellsand incubated at room temperature for 2 hrs followed by the above washsteps. The binding signal was determined based on HRP activity usingAmplex Red Peroxidase Assay (Invitrogen, CA) according to themanufacturer's instructions. The experiments were done in triplicate.Minimal binding signals were observed in the negative controls includingbinding of precomplexed unit to wells without glycans and binding of theantibodies alone to the wells with glycans. The binding parameters,cooperativity (n) and apparent binding constant (Kd′), for H2 HA-glycanbinding were calculated by fitting the average signal value (from thetriplicate analysis) and the HA concentration to the linearized form ofthe Hill equation:

${{\log\left( \frac{y}{1 - y} \right)} = {{n*{\log\left( \lbrack{HA}\rbrack \right)}} - {\log\left( K_{d}^{\prime} \right)}}},$where y is the fractional saturation (average binding signal/maximumobserved binding signal). The theoretical y values calculated using theHill equation

$y = \frac{\lbrack{HA}\rbrack^{n}}{\lbrack{HA}\rbrack^{n} + K_{d}^{\prime}}$(for the set of n and Kd′ parameters) were plotted against the varyingconcentration of HA to obtain the binding curves for representativehuman (6′SLN-LN) and avian receptors (3′SLN-LN) shown in FIG. 10.Human Respiratory Tissue Binding Assay

Formalin fixed and paraffin embedded normal human tracheal and alveolartissue sections were purchased from US Biological and US Biomax,respectively. Tissue sections were incubated for 30 minutes in ahybridization oven at 60° C. to melt the paraffin. Excess paraffin wasremoved by multiple washes in xlyene. Sections were subsequentlyrehydrated in a series of ethanol washes. In order to preventnonspecific binding, sections were pre-blocked with 1% BSA in PBS for 30minutes at room temperature (RT). For the generation of HA-antibodyprecomplexes, the histidine tagged purified recombinant HAs (Alb58,CkPA04, LS and TLS) were incubated with primary antibody against his tag(mouse anti 6×His tag, Abcam) and secondary (Alexa Fluor 488 goat antimouse IgG, Invitrogen) antibody in a ratio of 4:2:1 respectively for 20minutes on ice. Tissue sections were incubated with the HA-antibodyprecomplexed unit, diluted to different final concentrations in 1%BSA-PBS, for 3 hours at RT. Sections were then incubated with propidiumiodide to counterstain the nuclei (Invitrogen; 1:100 in TBST) for 20minutes at RT. After thorough washing, sections were mounted andanalyzed using a Zeiss LSM510 laser scanning confocal microscope.

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Example 2 Testing Inventive Binding Agents in an Animal Host

As described herein, the present invention encompasses the recognitionthat the use of animal hosts (e.g., ferrets) for the study oftransmission of virus may provide a reliable indicator of human virustransmission. Similarly, the present invention encompasses therecognition that the use of animal hosts (e.g., ferrets) treated withinventive binding agents (e.g., HA polypeptides) for the study oftransmission of virus may provide a reliable indicator of the efficacyof such inventive binding agents for prevention or treatment of virus ina human host.

The present Example describes a virus transmission assay that can beused in the presence or absence of inventive binding agents to determineviral transmission in a suitable animal model. Animal hosts, e.g.,ferrets, can be housed in adjacent cages that prevent direct andindirect contact between animals. However, these housing conditionsallow the spread of influenza virus through the air. A first portion ofthe animals are inoculated via methods known in the art, e.g.,intranasally, with an effective amount of virus (“inoculated animals”).Naïve animals can then be introduced into cages adjacent to theinoculated animals one, two, three or more days later.

Animals used in the study can be killed at any time one, two, three ormore days post-inoculation or transmission for analysis. Suitableanalysis for virus transmission studies can include, but is not limitedto determination of infectious virus titers (e.g., by nasal washes),observation of physical symptoms in the animals (e.g., lethargy,anorexia, rhinorrhea, sneezing, high fever, and/or death),immunohistochemical analysis of respiratory tissues, among others.

The virus transmission assay described above can also incorporate thetreatment of the animal host with an inventive binding agent describedherein before, during or after inoculation or transmission of virus.Analytic methods described herein are then used to determine theefficacy of the binding agent(s) in blocking transmission and/orinfection of the animal host with the virus.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

We claim:
 1. An engineered influenza hemagglutinin (HA) polypeptide thatpossesses at least 90% overall sequence identity with a reference HApolypeptide of SEQ ID NO: 1, which engineered HA polypeptidecharacterized in that its amino acid sequence includes: an amino acidresidue (“Residue 137”) at a position corresponding to position 137 ofSEQ ID NO:1 that is selected from the group consisting of arginine,lysine, glutamine, methionine and histidine; and an amino acid residue(“Residue 193”) at a position corresponding to position 193 of SEQ IDNO:1 that is selected from the group consisting of alanine, asparticacid, glutamic acid, leucine, isoleucine, methionine, serine, threonine,cysteine, and valine; and an amino acid residue (“Residue 226”) at aposition corresponding to position 226 of SEQ ID NO:1 that is selectedfrom the group consisting of alanine, cysteine, glycine, isoleucine,leucine, methionine, phenylalanine, proline, tryptophan and valine; andan amino acid residue (“Residue 228”) at a position corresponding toposition 228 of SEQ ID NO:1 that is selected from the group consistingof arginine, asparagine, aspartic acid, glutamic acid, glutamine,histidine, lysine, serine, glycine, threonine, and tyrosine.
 2. Theengineered HA polypeptide of claim 1, wherein the Residue 137 isselected from arginine, lysine, glutamine, and methionine.
 3. Theengineered HA polypeptide of claim 1, wherein the Residue 193 isselected from the group consisting of alanine, glutamic acid, threonine,cysteine, methionine, valine, and serine.
 4. The engineered HApolypeptide of claim 1, wherein the Residue 226 is selected from thegroup consisting of leucine, isoleucine and valine.
 5. The engineered HApolypeptide of claim 1, wherein the Residue 228 is selected from thegroup consisting of arginine, asparagine, serine, glycine, andthreonine.
 6. An engineered HA polypeptide characterized in that itsamino acid sequence includes the Residue 137 is arginine; Residue 193 isthreonine; Residue 226 is leucine; and Residue 228 is serine.
 7. Theengineered HA polypeptide of claim 1, wherein the HA polypeptide is anH2 polypeptide.
 8. The engineered HA polypeptide of claim 1,characterized in that the HA polypeptide binds to umbrella-topologyglycans with high affinity.
 9. The engineered HA polypeptide of claim 8,wherein the HA polypeptide binds the umbrella topology glycans with anaffinity comparable to that of a wild-type HA of the same subtype thatis human transmissible.
 10. The engineered HA polypeptide of claim 8,wherein the HA polypeptide binds the umbrella-topology glycans with anaffinity that is at least 50% that of a wild-type HA of the same subtypethat is human transmissible.
 11. The engineered HA polypeptide of claim8, wherein the HA polypeptide binds the umbrella-topology glycans withan affinity that is at least 70% that of a wild-type HA of the samesubtype that is human transmissible.
 12. The engineered HA polypeptideof claim 8, wherein the HA polypeptide binds the umbrella-topologyglycans with an affinity that is at least 90% that of a wild-type HA ofthe same subtype that is human transmissible.
 13. The engineered HApolypeptide of claim 8, wherein the HA polypeptide binds theumbrella-topology glycans with an affinity that is at least 100% that ofa wild-type HA of the same subtype that is human transmissible.
 14. Theengineered HA polypeptide of claim 1, wherein the HA polypeptide bindsto umbrella-topology glycans preferentially as compared withcone-topology glycans.
 15. The engineered HA polypeptide of any claim 1,wherein the HA polypeptide binds to umbrella-topology glycans vscone-topology glycans with a relative affinity of at least
 2. 16. Theengineered HA polypeptide of claim 1, wherein the HA polypeptide bindsto umbrella-topology glycans vs cone-topology glycans with a relativeaffinity of at least
 4. 17. The engineered HA polypeptide of claim 1,wherein the HA polypeptide binds to umbrella-topology glycans vscone-topology glycans with a relative affinity of at least
 10. 18. Theengineered HA polypeptide of claim 1, wherein the HA polypeptide bindsto umbrella-topology glycans vs cone-topology glycans with a relativeaffinity of at least
 20. 19. The engineered HA polypeptide of claim 1,characterized in that the HA polypeptide binds to cone-topology glycanswith low affinity.
 20. The engineered HA polypeptide of claim 1, whereinthe HA polypeptide binds to umbrella-topology glycans with a Kd′ ofabout 200 pM or less.
 21. The engineered HA polypeptide of claim 19,wherein the HA polypeptide binds to umbrella-topology glycans with a Kd′of about 100 pM or less.
 22. The engineered HA polypeptide of claim 19,wherein the HA polypeptide binds to umbrella-topology glycans with a Kd′of about 50 pM or less.
 23. The engineered HA polypeptide of claim 19,wherein the HA polypeptide binds to umbrella-topology glycans with a Kd′of about 20 pM or less.
 24. The engineered HA polypeptide of claim 1,wherein the HA polypeptide binds to cone-topology glycans with a Kd′ ofabout 100 pM or more.
 25. The engineered HA polypeptide of claim 23,wherein the HA polypeptide binds to cone-topology glycans with a Kd′ ofabout 200 pM or more.
 26. The engineered HA polypeptide of claim 23,wherein the HA polypeptide binds to cone-topology glycans with a Kd′ ofabout 500 pM or more.
 27. The engineered HA polypeptide of claim 23,wherein the HA polypeptide binds to cone-topology glycans with a Kd′ ofabout 1 nM or more.
 28. The engineered HA polypeptide of claim 1,characterized in that the HA polypeptide binds to umbrella-topologyglycans with high affinity and binds to cone-topology glycans with lowaffinity.
 29. The engineered HA polypeptide of claim 28, wherein the HApolypeptide binds to umbrella-topology glycans with a Kd′ of about 200pM or less and binds to cone-topology glycans with a Kd′ of about 200 pMor more.
 30. A method of inducing an immune response by administering acomposition comprising an engineered HA polypeptide of claim
 1. 31. Animmunogenic composition comprising the engineered HA polypeptide ofclaim
 1. 32. The immunogenic composition of claim 31, wherein theimmunogenic composition comprises a live attenuated virus.
 33. Theimmunogenic composition of claim 31, wherein the immunogenic compositioncomprises virus-like particles.
 34. The immunogenic composition of claim31, wherein the immunogenic composition is a subunit immunogenic. 35.The immunogenic composition of claim 31, further comprising an adjuvant.36. An engineered HA polypeptide wherein the improvement comprises thepresence of an amino acid residue (“Residue 137”) at a positioncorresponding to position 137 of SEQ ID NO:1 that is selected from thegroup consisting of arginine, lysine, glutamine, methionine andhistidine; and an amino acid residue (“Residue 193”) at a positioncorresponding to position 193 of SEQ ID NO:1 that is selected from thegroup consisting of alanine, aspartic acid, glutamic acid, leucine,isoleucine, methionine, serine, threonine, cysteine, and valine; and anamino acid residue (“Residue 226”) at a position corresponding toposition 226 of SEQ ID NO:1 that is selected from the group consistingof alanine, cysteine, glycine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan and valine; and an amino acid residue(“Residue 228”) at a position corresponding to position 228 of SEQ IDNO:1 that is selected from the group consisting of arginine, asparagine,aspartic acid, glutamic acid, glutamine, histidine, lysine, serine,glycine, threonine, and tyrosine.
 37. An engineered HA polypeptidewherein the improvement comprises the presence of an arginine at Residue137, a threonine at Residue 193, a leucine at Residue 226 and a serineat Residue
 228. 38. The engineered HA polypeptide of claim 1, whereinthe polypeptide shows at least 91% overall sequence identity with areference HA polypeptide of SEQ ID NO:1.
 39. The engineered HApolypeptide of claim 1, wherein the polypeptide shows at least 92%overall sequence identity with a reference HA polypeptide of SEQ IDNO:1.
 40. The engineered HA polypeptide of claim 1, wherein thepolypeptide shows at least 93% overall sequence identity with areference HA polypeptide of SEQ ID NO:1.
 41. The engineered HApolypeptide of claim 1, wherein the polypeptide shows at least 94%overall sequence identity with a reference HA polypeptide of SEQ IDNO:1.
 42. The engineered HA polypeptide of claim 1, wherein thepolypeptide shows at least 95% overall sequence identity with areference HA polypeptide of SEQ ID NO:1.
 43. The engineered HApolypeptide of claim 1, wherein the polypeptide shows at least 96%overall sequence identity with a reference HA polypeptide of SEQ IDNO:1.
 44. The engineered HA polypeptide of claim 1, wherein thepolypeptide shows at least 97% overall sequence identity with areference HA polypeptide of SEQ ID NO:1.
 45. The engineered HApolypeptide of claim 1, wherein the polypeptide shows at least 98%overall sequence identity with a reference HA polypeptide of SEQ IDNO:1.
 46. The engineered HA polypeptide of claim 1, wherein thepolypeptide shows at least 99% overall sequence identity with areference HA polypeptide of SEQ ID NO:1.